Blockchain Papers

Faith T. Campbell

Invasive exotic or alien species cause enormous damage to the natural environment. Indeed, 49% of all imperiled species in the United States are threatened in part by alien species (Wilcove et al. 1998). The toll may be even higher: Additional plant species have been harmed by alien species but have not yet been designated as imperiled (e.g., the American chestnut, Fraser fir, American elm, flowering dogwood). Losses in the agriculture, forestry, and other sectors of the US economy and control costs associated with invasive alien species total $137 billion annually. Approximately $80 billion of that amount is associated with alien plants, insects, and disease pathogens subject to phytosanitary regulations (Pimentel et al. 1999), which are the subject of this article. Because attempts to eradicate established invasive alien species have met with little success, scientists recommend that governments put the highest priority on preventing introduction of such species. This article examines US policies governing the structure and implementation of prevention programs aimed at alien plants and those insects and disease pathogens considered to be plant pests. Whereas many invasive plants have been imported deliberately, insects and fungal pathogens usually hitch a ride with imported cargo. As Chris Bright noted during a presentation at the Worldwatch Institute on 8 October 1998, international trade “leaks” exotic species. Many others also have predicted that severe damage would follow introduction of insects and fungal pathogens from forests in Europe, Mexico, Asia, or other regions that trade heavily with the United States (Forest Service 1991, 1998, Campbell and Schlarbaum 1994, Niemela and Mattson 1996, APHIS 1998c, 1998d). Some types of imports are particularly likely to transport hitchhikers. Live plants imported for horticultural use, for example, have been called a “uniquely efficient pest introduction pathway” (Regelbrugge 1998). Between 45% and 86% of various categories of pests introduced to California in recent decades came in on horticultural imports (OTA 1993). Among forest pests that were most likely introduced on imported live plants are dogwood anthracnose (Discula destructiva Redlin) (Campbell and Schlarbaum1994), Melampsora fungus on larch and poplar, and pine pitch canker (Fusarium subglutinans f. sp. pini) on Monterrey pine (Pinus radiata) (Haleamau 1998). Like live plants, “green” wood—raw or unprocessed wood—is a high-risk import. Crates, pallets, spools for cable, and other packaging are often made from low-quality, pest-damaged wood. When not in use, these items are usually piled in the open air, thus facilitating pests' establishment (APHIS 1998d). As US imports have grown—by more than 50% just since 1990 (GAO 1997)—so too has the quantity of solid wood packaging in which they are shipped, thus presenting more opportunities for exotic species to travel to North America. Another wood pathway for pests is through imported logs, unprocessed lumber, and wood chips. Those US imports once came almost exclusively from Canada, which is a low-risk source because forests in these two nations are contiguous. However, beginning in the 1990s, US wood processors have imported increasing quantities of green wood from geographically separate areas that harbor pests inimical to US forests. Although Canada still supplies more than 70% of US wood imports, Mexico supplies more than 3%, China more than 2%, Chile nearly 2%, and New Zealand about 0.6%; the remainder comes from tropical countries. These imports are expected to increase in the future now that a court injunction limiting imports has been lifted. Some knowledgeable observers, including scientists working with horticultural and forest pests (La Fage and Williams 1979, Niemela and Mattson 1996), as well as the National Plant Board (National Plant Board 1999), have concluded that the phytosanitary safeguards set out by the US Department of Agriculture (USDA) are inadequate to prevent introductions at current trade levels. According to the National Plant Board, “Recent breaches of the APHIS-PPQ [Animal and Plant Health Inspection Service, Plant Protection and Quarantine] safeguarding system that led to entry of dangerous invasive plant pests in the U.S. have raised concerns that current organizational policies and procedures are inadequate to execute Agency functions…. Clearly, the current safeguarding system cannot meet the changes thrust upon it by rapidly transforming global circumstances” (National Plant Board 1999, p. 40). Yet with imports rising, the danger of alien organisms reaching America is increasing, and phytosanitary controls would have to be tightened commensurately to ensure the same level of protection. APHIS has begun to strengthen its regulations for wood packing (APHIS 1998e, 1999a) and lumber from Mexico (APHIS 1999b); both of these rulemakings are still in process. Nonetheless, it has fought in court against scientists who argue that regulations should be strengthened also for logs and lumber from New Zealand and Chile; it has rejected similar pleas from the Oregon Department of Agriculture for regulation strengthening. Moreover, the USDA has relaxed controls for certain horticultural imports. To be “science based,” a phytosanitary program should reflect the serious threat posed by exotic species to agriculture, horticulture, and forestry, and to the myriad natural ecosystems and biotic communities found from Alaska to Florida and from Maine to Hawaii. It should also reflect practical realities. First, ecologists realize that their knowledge of the millions of insects, fungi, and disease pathogens living in the habitats of US trading partners is limited. Indeed, probably less than 5% of the Earth's fungi have even been named by science; fewer still have been studied enough to determine their ecological role. Scientists know more about exotic insects and plants, but even among these groups are tens of thousands of species that have not been well studied. Given these large information gaps, it may well be impossible for scientists to predict which foreign species might cause devastating damage if introduced to a new ecosystem. Second, biological invasions are usually irreversible, given current scientific knowledge, limited funding, and burgeoning pest problems. In other words, it is far easier—and less expensive—to prevent an invasion than to combat one. It follows that a sound phytosanitary program should seek to bar entry to all exotic organisms except those that have been evaluated and determined to be very unlikely to be invasive. Two slogans express this goal: “If in doubt, keep it out” and “Guilty until proven innocent.” To put this objective into practice, the responsible government agency—USDA's APHIS—could combine technological treatments with stringent regulatory requirements that importers act to minimize the likelihood that living, unapproved insects, fungal pathogens, viruses, or weeds will hitchhike to this country on imported goods. This approach obviates several problems. First, it reduces the burden on port inspectors, who would otherwise have to search millions of shipments for tiny, even microscopic, organisms. Second, it reduces the risk that an erroneous assessment of the potential impacts of an organism will result in a decision to allow entry of a species that turns out to be highly damaging. An appropriately serious phytosanitary program should also seek out and utilize all available and emerging information on pests, pathways, and exclusion or mitigation technologies, and rapidly adapt regulations to reflect that new information. However, USDA officials have rejected the “If in doubt, keep it out” policy (Isi Siddiqui [US Deputy Under Secretary of Agriculture], personal communication, 1998), finding it incompatible with trade promotion; APHIS is now trying to “balance” protection and trade promotion. Thus, rising trade volume is not the sole reason that the threat of introduction of harmful bioinvaders is growing. Although USDA policy that proves ineffective can be changed through domestic political channels, pro-trade policies now have the force of international law through US adherence to global trade rules enforced by the World Trade Organization (WTO). Thus, if these policies do result in too high a risk of introduction of pests, international treaties adopted by 135 countries must be changed—a daunting challenge. Phytosanitary measures are no longer adopted unilaterally in response to a country's perception of risk. Phytosanitary safeguards are now subject to international standards and review. In 1994 more than 100 countries concluded the Uruguay Round of the General Agreement on Tariffs and Trade by forming the World Trade Organization, which sets the rules for international trade. To prevent member nations from using phytosanitary safeguards to protect domestic agricultural producers from foreign competition, the signatories simultaneously adopted the Agreement on the Application of Sanitary and Phytosanitary Measures (SPS agreement; see www.wto.org). In November 1997, the 46-year-old International Plant Protection Convention (IPPC), which was formed to control plant pests and diseases and prevent their spread across national borders, was revised to bring it into compliance with the SPS agreement. (The IPPC revisions have not yet come into force, however, because too few nations have ratified the changes.) Under the SPS agreement and revised IPPC, countries may impose trade restrictions to the extent necessary to protect human, animal, or plant health—including wild fauna and flora—as long as they apply the same standards for protection across the board, domestically as well as internationally. Phytosanitary measures must be based on scientific principles and supported by scientific evidence, as documented in a risk assessment. When scientific information is insufficient to meet the requirements for risk assessment laid out in the SPS agreement, a country may adopt a provisional regulation while seeking more information. Taking into account available scientific evidence, the risk assessment should address processing or production methods; inspection and sampling methods; prevalence of the target organism—or the existence of pest- or disease-free areas—in the exporting and importing countries; ecological and environmental conditions; and quarantine or treatment measures that could control any pest that is introduced. A risk assessment should also take into consideration the potential damages from introduction of a pest or disease (such as losses in production or sales and the cost of control or eradication) and the cost-effectiveness of alternative approaches to limiting risks. The SPS agreement states that phytosanitary regulations should not be more restrictive than necessary to achieve a country's chosen level of protection—which could be set at zero risk—and that the process of their promulgation must be transparent. Finally, the SPS agreement grants developing countries more time to comply with international phytosanitary standards and calls on WTO member countries to give them technical assistance. Countries that believe their exports have been discriminated against may challenge the importing country's phytosanitary protections as nontariff barriers to trade; if the challenger wins its case, the importer must either revise the phytosanitary regulations or pay a penalty. The penalty can be substantial: For example, after the European Communities (EC) refused to accept a WTO Appellate Body ruling allowing entry of US beef from cattle treated with growth hormones, the United States imposed a $116.8 million tariff on various European food exports (Williams 1999). The SPS agreement is only now being interpreted—often through official decisions of WTO dispute settlement bodies—thus many questions remain to be answered about what the agreement allows. USDA is wrestling also with questions about how it will apply certain provisions. For example, what level of protection should the United States strive to maintain? Which agencies, in addition to APHIS, should weigh in on this policy? Because any pests introduced as a result of APHIS policies will affect everyone in the United States, as well as the environment, all of the issues surrounding the SPS agreement should be the subject of broad discussion. Two topics in particular—determination of an appropriate level of protection and “quarantine pests”—should be part of that discussion. As mentioned above, each country has the right to set its own level of protection, even if that level is zero risk. However, a country must consistently apply comparable levels of protection in comparable situations; a further limitation to this right to determine an appropriate level of protection, according to the WTO Appellate Body, is that the country is obligated to demonstrate a scientific justification for the selected level of risk (Victor 1999). The consistency requirement exposes any phytosanitary safeguard to comparison with other domestic or international statutes aimed at managing a comparable risk or—importantly—to comparison with the absence of such statutes. A gap in the protection provided by these statutes could be cited as proof that a stringent phytosanitary regulation is inconsistent with existing standards. Indeed, the Appellate Body ruled against Australia when that country banned imports of salmon largely because the sanitary measure for salmon imports was more stringent than that for other fish imports judged—by the WTO dispute panel—to pose an equal or greater risk (WTO AB-1998-5). For stringent phytosanitary safeguards to survive a challenge under the SPS agreement, then, countries must close any loopholes in regulations governing comparable situations. Of course, tightening these statutes takes time and may prove difficult because of political pressures. Until these steps are taken, however, differences in a country's approach to various risks to health and to the environment might be challenged as or are to be (WTO The SPS dispute have also ruled that a country's of its level of risk be supported by a risk that assessment must be even scientists have little to predict which foreign species may prove when introduced to a new environment. In the from US the dispute supported by the Appellate Body, noted that the scientific on which the European Communities based their level of risk were in the of they not as the Appellate Body they the potential of the at levels found more or when the are for growth in (WTO p. this level of to a risk assessment for potential plant pests might restrictions on imports by both that a fungus has the potential to damage in the importing country and that the fungus might be on an imported The risk assessment would probably have to address each species or to logs for the Appellate Body that is not is a of A risk the the of or (WTO p. Although the Appellate Body imposed it that the not have to be must it prove that the damage would any (WTO AB-1998-5). Given current levels of scientific knowledge and funding, can APHIS be expected to the likelihood or that any of the of insects, fungi, or plant of which could hitch a ride on any from any of US trading any of the agricultural or natural ecosystems of the United Moreover, the Appellate Body not the that many other potential pests cannot be evaluated at because they are either to or or in their APHIS can apply phytosanitary an organism for control must have been designated a “quarantine must be based on a risk the in and scientific just In practice, for example, APHIS might well be to predict that certain insects on imports of pose a threat to in do scientists have information to how those insects might in natural to the potential for invasion by other species of or pathogens on the same It is that APHIS can these the time to risk that imports of to pose severe pest risks can for several APHIS can the process and impose new For example, APHIS in that it considered solid wood packing to be of the pest of all wood imports (APHIS it its risk assessment regulations only in October APHIS the in The only a country can the to the and its with such is through of provisional measures under the of article In ruling on the US challenge regulations on imports of and the Appellate Body that a measure cannot as provisional the country is seeking the information and will the provisional measure a (Victor 1999). The is in the many in which APHIS with it must either to allow the organism with invasive potential into the it will not prove to impose a provisional on its and to trying to the standards of the SPS agreement. The to for the of information on species may well be to on of it has been determined that pests may be introduced a certain of or its country of APHIS should to the available to that or as as to insects, fungi, and the SPS agreement allow a country to adopt such an safeguard on the of a risk assessment that examines a of pests that might be associated with that and not Although pathway regulation has not yet been at in a the on in the of the agreement and in Appellate Body that such an approach would be found when an organism has been designated a “quarantine the still phytosanitary to ensure that their phytosanitary requirements cause the to trade. In other words, of the most measure to against or a country must accept risk of that and adopt those measures that a level of protection. For example, in the of logs and lumber imported from New Zealand and APHIS the less of alternative measures in to the burden on APHIS with that treatments would be more than with in preventing pest its of by that is to the agricultural and its forest from exotic plant pests, but it also has the ensure international only to the to meet plant protection of plant pest risk to a To this would be considered trade restrictions under and subject to challenge by other trading partners in the World Trade (APHIS p. The SPS agreement and revised IPPC the right of the United States to protect from introduction of the thousands of species of pests and weeds that are natural ecosystems in the country insects, see Niemela and Mattson 1996, APHIS for plants, see and the Plant at more organisms to these species into the country several The the species to the new imports may a in an not and the imported organisms may a that is to control than established from the New Zealand see et al. 1999). the SPS agreement and IPPC allow a country to phytosanitary barriers for pests in the country only when the species is not and an official control program the or when the introduced organism from its in the United States in a that the potential to cause greater which as an control will affect the types and of established pests and weeds against which APHIS can protect US ecosystems from imports. however, the to control programs for more than a few species. A of could for phytosanitary controls at US borders, provided that regulatory and programs are considered control Given the that could result from allowing introductions of established it would be appropriate to all in the of which of are to be official control As noted above, the SPS agreement also governments to of an established pest when the new in from in the However, phytosanitary have been to in their attempts to determine such that can have serious the spread of of disease by several fungi in the which when phytosanitary imports of logs, based on their that the disease only that was in their countries. APHIS policies and procedures to be which may the threat from of the SPS agreement. For example, according to the National Plant Board, APHIS-PPQ risk assessment process not the in the it on which or are Many of the process are (National Plant Board 1999). In response to this APHIS has a of its risk assessment October 1999, the a in the seeking on the and on November 1999, it a the an in risk on a process and noted in APHIS the for with the process to ensure that no is also noted that APHIS too on the of an introduction and too little on the of such an introduction 1999). APHIS is also a to and existing international standards and the current of the APHIS to a with of changes in ensure and The SPS agreement was almost by in trade and not in the new of invasion Under the it is not that the SPS agreement phytosanitary safeguards and the danger of harmful The danger from to heavily on risk when the are the priority to trade protection from biological invasion not the of species and biological subject to its on and a few species than on preventing any introductions the of the differences in risk of associated with shipments from as to producers A for American scientists to the issues raised in this article and to for changes in policies that to pest exclusion In US an Invasive and it with developing for US programs to prevent and to The Invasive should the SPS agreement and IPPC allow countries to adopt phytosanitary programs to the structure in In the APHIS can its pest exclusion program the imposed by the SPS agreement and IPPC by zero which it should appropriate level of protection. Although this new would not “If in doubt, keep it out” or “Guilty until proven as the in it would the to be as as when for example, which organisms should be considered “quarantine As above, APHIS is to its risk assessment assessment may be an appropriate for to set for search and on pests, but the process is too and too to to a scientific for an exclusion

Wenbo Mao

Let n be a large composite number. Without factoring n, the computation of a 2 t (mod n)given a, t with gcd(a# n) = 1 and t!n can be done in t squarings modulo n.For t n (e.g., n?2 1024 and t!2 100 ), no lower complexity than t squarings is known to fulfill this task. Rivest et al suggested to use such constructions as good candidates for realising timed-release crypto problems. We argue the necessity for a zero-knowledge proof of the correctness of such constructions and propose the first practically efficient protocol for a realisation. Our protocol proves, in log 2 t standard crypto operations, the correctness of (a e ) 2 t (mod n) with respect to a e where e is an RSA encryption exponent. With such a proof, a Timed-release Encryption of a message M can be given as a 2 t M (mod n) with the assertion that the correct decryption of the RSA ciphertext M e (mod n) can be obtained by performing t squarings modulo n starting from a. Timed-release RSA signatures can be constructed analogously. Keywords Timed-release cryptography, Time-lock puzzles, Non-parallelisability, Efficient zero-knowledge protocols. 1

Tzafrir Cohen, Joe Kilian, Erez Petrank

No abstract is available for this record.

Glenn Durfee, Matthew Franklin

Digital content distribution systems will enable business models in the near future that cannot be predicted today. In this paper, we identify a new security problem that can be crucial to this enablement. The problem arises from the conflicting privacy and integrity goals of middlemen in digital distribution chains. Our solution is a novel system design that incorporates obfuscated digital contracts, semi-trusted contract certifiers, and zero-knowledge proofs of arithmetic relations. Our implementation and timing experiments demonstrate that our solution is practical and efficient.

Joan Boyar, Ivan Damgård, René Peralta

No abstract is available for this record.

Charles F. Minto, Thomas W. Schnider, Timothy G. Short, Keith M. Gregg · 6 authors

Click on the links below to access all the ArticlePlus for this article.Please note that ArticlePlus files may launch a viewer application outside of your web browser.DRUG interactions are the basis of anesthetic practice. For example, induction of anesthesia may consist of intravenous administration of a benzodiazepine before induction, a hypnotic to achieve loss of consciousness, and an opioid to blunt the response to noxious stimulation. Similarly, anesthesia often is maintained with a combination of a hypnotic (e.g. , propofol, isoflurane) and an analgesic (e.g. , fentanyl, nitrous oxide). Anesthetic drugs are often combined because they interact synergistically to create the anesthetized state.Pharmacodynamic drug interactions are typically described using mathematical models. The basic model is that of an isobole. Isoboles are iso-effect curves, curves that show dose combinations that result in equal effect. 1The combination of two doses (d1and d2) can be represented by a point on a graph, the axes of which are the dose axes of the individual drugs (fig. 1). The isobole connects isoeffective doses of the two drugs when administered alone, D1and D2. If the isobole is straight (fig. 1A), then the relation is additive. If the isobole bows toward the origin (fig. 1B), then smaller amounts of both drugs are needed to produce the drug effect when administered together, so the relation is supraadditive or synergistic. If the isobole bows away from the origin (fig. 1C), then greater amounts of both drugs are needed to produce the drug effect when administered together, so the relation is infraadditive. In table 1we propose a set of criteria that pharmacodynamic models of drug interactions should meet. In this article we propose an interaction model that meets these criteria, based on response-surface methodology. Response surfaces are a powerful statistical methodology for estimating and interpreting the response of a dependent variable to multiple inputs. 2Response-surface methodology is used for two principal purposes; to provide a description of the response pattern in the region of the observations studied and to assist in finding the region in which the optimal response occurs. Our model is a straightforward extension of the sigmoidal concentration–response relation for individual drugs. We test the proposed model using data from a study of the interaction of midazolam, propofol, and alfentanil with loss of consciousness. 3This article only considers pharmacodynamic interactions, the type of interaction most relevant to the practice of anesthesia. Pharmacokinetic interactions are entirely different and will not be considered. Appendix 1 (which can be found on the Anesthesiology Web site at www.anesthesiology.com) reviews several commonly used pharmacodynamic models of drug interactions and shows areas in which existing models fail to meet the criteria in table 1.The effects of individual drugs are often modeled by relating drug effect (E) to drug concentration (C) using a sigmoid model:where E0is the baseline effect when no drug is present, Emaxis the peak drug effect, C50is the concentration associated with 50% drug effect, and γ is a “sigmoidicity factor” that determines the steepness of the relation.This relation is shown graphically in figure 2. The concentration term often is defined as the concentration at the site of drug effect, but the model can be generalized to any measure of exposure (e.g. , dose, plasma concentration, or area under the curve). For models of probability, such as the probability of moving in response to surgical incision, E0is 0 and Emaxis the maximal probability (usually assumed to be 1). Dividing the numerator and denominator of equation 1by C50γ, we obtain an alternate form:In this model, concentration has been normalized to the concentration that results in 50% of maximal drug effect. This is a natural way to think about drug concentration—as a fraction of some measure of potency. For example, anesthesiologists are accustomed to thinking about volatile anesthetics in terms of minimum alveolar concentration (MAC), rather than in absolute concentration terms. This is precisely the concept of normalizing drug concentration to potency.The basic concept of our proposed interaction model is simple. Consider two drugs, each of which has a sigmoidal concentration–response relation. We will think of any given ratio (i.e. , B/(A + B), called θ herein) of the two drugs as behaving as a new drug. This new drug, which is actually a fixed ratio of the two drugs, has its own sigmoidal concentration–response relation, as shown in figure 3. This is the basic premise of our interaction model. The mathematics are simply an extension of the model for a single drug to a model that considers each ratio of two drugs as a drug in its own right. We will express the concentrations of drugs A and B as [A] and [B]. As suggested by equation 2, we must first normalize each drug to its potency, C50, and express the results in units (U) of potency. where UAis the normalized concentration of drug A, and UBis the normalized concentration of drug B. We can define a family of “drugs,” each being a unique ratio of UAand UB. Each drug will be defined in terms of θ, where θ is defined as By definition, θ ranges from 0 (drug A only) to 1 (drug B only). The “drug concentration” is simply UA+ UB. We can extend equation 2to describe the concentration–response relation for any ratio, θ, of the two drugs in combination:where θ is the ratio of the two drugs, the drug concentration is UA+ UB, γ(θ) is the steepness of the concentration–response relation at ratio θ, U50(θ) is the number of units (U) associated with 50% of maximum effect at ratio θ, and Emax(θ) is the maximum possible drug effect at ratio θ. Because Emax, C50, and γ in equation 2have been replaced by functions of θ, each ratio has the potential to have its own Emax, C50, and γ. This allows each ratio of drug A and drug B to behave as its own drug, with its own sigmoidal concentration–response relation, which is the basic premise of the model.The term “U50(θ)” is the potency of the drug combination at ratio θ relative to the normalized potency of each drug by itself. This requires careful explanation. Let us assume that only drug A is present, in a concentration of C50,A. In this case, the drug effect is half of the maximal effect, UA= 1, UB= 0, θ= 0, and the drug concentration is UA+ UB= 1. Because we have 50% of the maximum drug effect, and 1 unit of drug, then the number of units associated with 50% drug effect when only drug A is present, U50(0), must be 1. Similarly, let us assume that only drug B is present and the concentration of drug B is C50,B. In this case, the drug effect is half of the maximal effect, UA= 0, UB= 1, θ= 1, and the drug concentration is UA+ UB= 1. Because we have 50% of the maximum drug effect, and 1 unit of drug, then the number of units associated with 50% drug effect when only drug B is present, U50(1), must again be 1. By definition, if only drug A or drug B is present, U50(θ) = 1.Now, let us assume that drug A and drug B both are present, each in exactly half of the concentration that would cause 50% of the drug effect when administered alone. In this case, UA= 0.5, UB= 0.5, θ= 0.5, and the drug concentration is UA+ UB= 1. If this causes 50% of maximum effect, then the drugs are simply additive at θ= 0.5, and U50(0.5) = 1. However, if this combination produces more than a half-maximal effect, then 1 unit of this combination, at θ= 0.5, is more potent than 1 unit of either drug alone (i.e. , synergistic). In this case, U50(0.5) < 1. Conversely, if this combination produces less than a half-maximal effect, then 1 unit of this combination, at θ= 0.5, is less potent than either drug alone (i.e. , infraadditive). In this case, U50(0.5) > 1. Thus, U50(θ) is the potency of the combination compared with the potency of either drug alone, which is 1 by definition.Thus, the units of U50(θ) are not concentration units, but rather the number of units, at ratio θ, associated with 50% of maximal drug effect. U50(θ) is 1 for θ= 0 and θ= 1. For all values of θ between 0 and 1 (i.e. , all possible ratios of the two drugs), U50(θ) assumes a value determined by the data. If this value is 1, then the interaction is additive at θ. If the value is less than 1, then the drug effect is synergistic at θ. If the value is greater than 1, then the interaction is antagonistic at θ.Figure 4shows the relation between a three-dimensional response surface and a conventional two-dimensional isobolographic analysis. The two-dimensional isobologram is a cut through the three-dimensional surface, generally taken at the 50% response level. In this particular example, synergy is evident in the three-dimensional model as a bowing of the surface toward the reader. This bowing causes the conventional isobologram to deviate toward the origin from the straight line of additivity. Much pharmacodynamic literature supports the sigmoid relation in equation 1, equation 2, and equation 5. There is only modest information specifying the functions Emax(θ), U50(θ), and γ(θ). Our choice is to use functions that are capable of taking a variety of shapes, so that good approximations to the true relations can be determined empirically. To provide these flexible functions we chose fourth-order polynomials of the form where f(θ) is Emax(θ), U50(θ), or γ(θ). The coefficients (β0, β1, β2, β3, β4) are model parameters that are either constrained by the model or estimated from the data. Fortunately, two of these terms, β0and β1, can be replaced by other terms already defined.We already defined the values Emax(θ), U50(θ), and γ(θ) when only drug A is present, Emax,A, U50,A, and γA, respectively. Note in equation 6that when θ= 0 (only drug A is present), f(0) =β0. Therefore, when f(θ) is Emax(θ), U50(θ), or γ(θ), β0must be Emax,A, U50,A, and γA, respectively.Similarly, we also defined the values Emax(θ), U50(θ), and γ(θ) when only drug B is present, Emax,B, U50,B, and γB, respectively. Referring again to equation 6, when θ= 1 (only drug B is present), f(1) =β0+β1+β2+β3+β4. We can rearrange this as β1= f(1) −β0−β2−β3−β4. Thus, when f(θ) is Emax(θ), U50(θ), or γ(θ), β1must be Emax,B− Emax,A−β2,Emax−β3,Emax−β4,Emax, U50,B− U50,A−β2, U50−β3,U50−β4,U50, or γB−γA−β2,γ−β4,γ, respectively.This permits us to develop models that incorporate the individual drug parameters for Emax(θ), U50(θ), and γ(θ) as functions of θ. The equation for Emax(θ), using the substitutions previously mentioned for β0and β1, is U50,Aand U50,B, [equivalent to U50(θ) and U50(1)], are both 1 by definition. Thus, when f(θ) = U50(θ), the values of β0and β1in equation 6are 1 and −β2−β3−β4, respectively. Therefore, the equation for potency as a function of θ can be simplified to Many isobolograms have a simple inward or outward curvature, which can be readily encompassed with a simple quadratic form of equation 8with just one coefficient:If β2,U50is 0, then the value of U50(θ) will be 1 for all values of θ. This means that the interaction will be additive. If β2,U50is a positive number, then U50(θ) will be less than 1 for all values of θ between 0 and 1. The effect is to magnify the term in equation 5, making it appear that there is more drug present. This will produce a greater than additive effect, i.e. , synergy. If β2,U50is a negative number, then U50(θ) will be greater than 1 for all values of θ between 0 and 1. This reduces the term in equation 5, making it appear that there is less drug present. This will produce a less than additive effect. This assumes that drugs A and B have the same maximal effect. It is possible for some approaches to synergy analysis to show apparent synergy if the maximal effects of drugs A and B are not identical, even if U50(θ) = 1 for all values of θ.The model for the steepness term, γ(θ), can similarly be described from equation 6, with appropriate substitutions for β0and β1. The resulting equation is Equations 6–10describe straight lines (simple additivity) when the coefficients (i.e. , β2, β3, β4) are 0. They are the equations for parabolas if the respective β2coefficient is nonzero, and β3and β4are 0. More complex shapes are generated when β3and β4are nonzero.Figure 5shows Emax, U50, and γ as functions of θ for the synergistic interaction seen in figure 4. Emaxand γ are constant, and thus have no interaction. U50is necessarily 1 at θ= 0 and θ= 1, but is less than one between these extremes. This increases the potency of the drugs when administered in combination, resulting in the synergy seen in figure 4. The model can be readily expanded to show the interaction of more than two drugs. In the case of three drugs (A, B, and C) the proportion of each drug present can be expressed by θA, θB, and θC, where We can define the ratio of three drugs from just two of these ratios because θA+θB+θC=1. For our purposes here, we will use θBand θC. We again assume that for any fixed value of θBand θC, there is a sigmoidal relation between concentration and response. Therefore, if the three drugs could be administered to the effect site in an exactly fixed proportion, they would show a sigmoidal total concentration–response relation, where the “concentration” was the sum of the three normalized concentrations. This is precisely the notion that underlies the two-drug model. The equation for the model the model the parameters of the sigmoidal relation, Emax, and U50, are functions of θBand θC. The functions and are described in Appendix (which can be found on the Anesthesiology Web site at The point is that of a as in equations when three drugs are present, the parameters of the sigmoidal relation are surfaces for functions of θBand θC. The response-surface model was in the by the and as a for for also the model for of at 3This is on to the using and for the use of our response-surface model, we data previously by data are also the Anesthesiology Web site relations intravenous doses of midazolam, propofol, and alfentanil administered and in combination in for as to the to administration and or alfentanil to peak effect an intravenous the combination being midazolam, it was administered before the other drugs. The doses of midazolam, propofol, and alfentanil used and the proportion of for each dose are shown in table 2. This data set was because it three two-drug combinations that could be used to In the data are for the number of and the of the it the to a interaction model, the of the proposed response-surface model. We assumed in the model that all when no drug was 0 by definition. In we assumed that each drug was capable of if administered in a a of equation and can be constrained to 0 and 1 with no interaction the Thus, the probability of for any combination is where UB, and the doses of midazolam, propofol, and respectively. The units are of each dose to cause in 50% of the based on equation the data for the single and combination of and the data for the single and combination of and alfentanil the data for the single and combination of and alfentanil and the data set for the and combinations modeled parameters and estimated using by the for all the response of the either 0 to or 1 to and is the probability of response to for each dose be expressed in as the sum of the natural of the of response in the and in the The of the coefficients to the model was by the coefficients one at a by the model ratio and by of probability of for each dose and The response surfaces for the interactions and the surface was used to the synergistic combinations of the and the interactions based on the from the analysis of the data The intravenous doses to achieve probability of in this for each drug alone, for each combination, and for the To the application of the response-surface model with the parameters of , , and used to the for midazolam, propofol, and respectively. This was as the concentration at the of the respective then used to the of effect of in the these synergistic doses of midazolam, propofol, and administered alone and in The the and the from in of the to in of the The of effect was using for for by and shapes of the response surfaces generated by the equations are not readily We used three-dimensional to the response surface for a variety of interactions between two drugs, by the model parameters of equation 5. interactions and antagonistic interactions between two and interactions between and to the data for all in the in to the used by , the of the data for and for the analysis of the three drug interactions are shown in table 3. and for the analysis of the data set are shown in table 4. There no in the of the three drugs, there drug interactions the The for each drug to be the of the combination being modeled and This is an Because the data for single administration was in the interaction the entirely determined from each drug alone. the response surfaces for each of the drug interactions synergy. The synergy in the model was not Appendix on the Anesthesiology Web site at This when all three drugs are present, there is not synergy that from the interactions of all three drugs. The surface is shown in figure The maximum in values for the combinations are represented by the of the three of the as a = = = The maximum in for the combination is found at the point of the surface, which is at and This in figure and where the point as a on the 5shows the doses for the and combinations for maximum synergy associated with probability of a of based on the parameters shown in figure The between probability of to probability of no for each combination was based on the shown in figure the synergistic combination administration of one the dose, this results in a in the for to alone is the drug of choice when the point is a of A of the and of other such as and is to the drug combinations for other our response-surface model for an additive interaction a synergistic interaction and an interaction It also shows the interaction between a and a a and a and a and an way to describe our model for two drugs, A and B, is that the drug A and the drug B there are two sigmoid 1). Our proposed model connects these two sigmoid curves functions of θ interactions are then as coefficients of the polynomials that the to each each value of θ can have its own Emax, C50, and the model assumes that the sigmoidal is for all values of θ. This concept of a fixed ratio of two drugs own is not each drug ratio to be to a single response could be described by a single two-dimensional concentration–response that when using combinations of combination should be as a new with individual rather than the of the individual use of functions to the parameters of drug A to drug B assumes that the response surface is and the basic model the parameters estimated in sigmoid the polynomials that U50(θ), Emax(θ), and γ(θ) are more The use of response functions to complex response surfaces is in However, models with the variable present at than the are not often used because it to the proposed a flexible model. of the response surface, or application of the could result in of parameters that provide a but when more an for the are not unique to this model and can be by the one is in the terms can be from the model. The statistical of the terms should be the models using the ratio In to of model such as of and the pattern of the model should also be by the response surface, and by the individual model parameters as functions of θ (e.g. , equations to that the pharmacodynamic parameters not to For it must be that U50(θ) and γ(θ) are positive in the of 0 1. In the case of three drugs, the surface should be as shown in figure a mathematical that if and only if the isobolograms are straight For this to be be with to θ. The case of γ(θ) is more is with to θ, there is if Emax(θ) and U50(θ) are also not equal to γB, there is no way to use our interaction model to test for defined by 1). we with of the surface, the description of an interaction as or antagonistic may be For example, a drug combination can be synergistic in and antagonistic in our the on drug interactions can be to simple such as or the The interaction has the potential to be and than about which to the relation, the should be to the response the surface one can the combination to produce the model is It no about the of interaction between the drugs. However, we assume that the concentration–response relation for each of the drugs is described by a pharmacodynamic model. We have not to describe interactions between drugs that and that we are not of any that our could not be combined with the more of model for each drug not have to be the sigmoid 1). For example, the model could be a or response The model could also be a response as seen with some the model can be any so as it has parameters that the individual models. Thus, the only is that the interaction model reduces to the model for drug A when θ= 0 and to the model for drug B when θ= the of response-surface can For example, the of effects the of data in a to the model parameters for one of the drugs. to specifying a administered function for the concentration–response relation is to use a as described by the use of flexible functions that are to example, a can be constrained to an they in the of antagonistic interactions, it can be to model additive and synergistic drug used our response-surface model to the drug interactions for the hypnotic point between midazolam, propofol, and on this a maximum effect of for all three drugs described the data the dose of and alfentanil the response to in of the dose of the response to in only of results not that alfentanil will or in response to a surgical they that doses of will response to in of the of our response-surface model to potential interactions of two or three drugs at the effect site We that these concentrations are based on information parameters and not possible interactions between the three drugs. this we to that the synergistic dose ratio is not necessarily if a hypnotic effect is the the three-dimensional may in and different used to study drug interactions, it not information that be by a of two-dimensional such as the isobologram of the response surface, of three-dimensional the response to concentration of one drug in the of fixed concentration of the other drug to one and the response to fixed concentration ratios of the two any of these a three-dimensional of the response surface can be if are In the case of three drugs, it is no possible to the response surface because it is a surface, the model parameters can be in three (fig. of using axes to be in the of the interactions between three or more drugs. the study of drug interactions in anesthesia has used isobolographic analysis or multiple approaches have In the multiple is so with described in Appendix 1 in the web with pharmacodynamic that it should be The application of response-surface methodology to the study of drug interactions has the potential to the of these models. We proposed a flexible model for drug interactions, which the relation between the concentrations of two or three drugs and drug effect. We our new model using previously data and that this model can also describe of interaction between an a a and an of response-surface methodology permits of the concentration–response relation and can be used to develop for optimal drug

Ran Canetti, Oded Goldreich, Shafi Goldwasser, Silvio Micali

We introduce the notion of Resettable Zero-Knowledge (rZK), a new security measure for cryptographic protocols which strengthens the classical notion of zero-knowledge. In essence, an rZK protocol is one that remains zero knowledge even if an adversary can interact with the prover many times, each time resetting the prover to its initial state and forcing it to use the same random tape. All known examples of zero-knowledge proofs and arguments are trivially breakable in this setting. Moreover, by definition, all zero-knowledge proofs of knowledge are breakable in this setting. Under general complexity assumptions, which hold for example if the Discrete Logarithm Problem is hard, we construct:

Jürgen U. Linder, Thomas Hoffmann, Ursula Kurz, Joachim E. Schultz

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His6-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu1681 and Ser1748 in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Gαs/GTP or forskolin, and P site inhibitors were only slightly effective. Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His6-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu1681 and Ser1748 in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Gαs/GTP or forskolin, and P site inhibitors were only slightly effective. nitrilotriacetic acid heterotrimeric guanine nucleotide-binding protein, GTPγS, guanosine 5′-O-(3-thiophosphate) guanylyl cyclase fromParamecium Adenylyl and guanylyl cyclases are key proteins in intracellular signaling of essentially all eukaryotic cells. In the freshwater protozoans Paramecium and Tetrahymena cAMP and cGMP levels depend on the ion composition of the extracellular milieu. Whereas adenylyl cyclase activity in vivo is stimulated by membrane hyperpolarization (1.Schultz J.E. Klumpp S. Benz R. Schürhof-Goeters W. Schmid A. Science. 1992; 255: 600-603Crossref PubMed Scopus (128) Google Scholar, 2.Schultz J.E. Schönborn C. FEBS Lett. 1994; 356: 322-326Crossref PubMed Scopus (17) Google Scholar), cGMP formation is enhanced by a depolarizing Ca2+ inward current (3.Schultz J.E. Pohl T. Klumpp S. Nature. 1986; 322: 271-273Crossref Scopus (48) Google Scholar). Recently, we reported on a group of guanylyl cyclases of 280 kDa that is present in the ciliates Paramecium and Tetrahymena but also in the genome of the malaria parasite Plasmodium (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). This novel signal transduction protein is bifunctional. It has a 155-kDa N-terminal P-type ATPase-like domain and a 115-kDa C-terminal guanylyl cyclase domain. Both are linked by a cytosolic loop of about 110 amino acids (see Fig. 1 A). Surprisingly, the guanylyl cyclase domain is topologically identical to mammalian adenylyl cyclases,i.e. it is composed of two pseudosymmetric modules of six putative transmembrane spans (M1 and M2) that serve as membrane anchors for the cytosolic, catalytic segments C1 and C2 (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). The sequences of both cytosolic subdomains are similar to each other and resemble the catalytic regions of metazoan adenylyl and, to a lesser extent, guanylyl cyclases. Sequence comparisons show that the order of the protozoan C1a- and C2-positioned loops is reversed compared with the corresponding mammalian adenylyl cyclase loops (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). Data based on the crystal structure of a soluble adenylyl cyclase type VC1a/type IIC2 chimera demonstrated that Lys938 and Asp1018 in the catalytic pocket of the C2 domain (adenylyl cyclase type II numbering) are responsible for ATP substrate specificity (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, 6.Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar). In theParamecium guanylyl cyclase, those two amino acids have evolved as Glu1681 and Ser1748 in the C1-positioned domain, i.e. specify GTP as a substrate, whereas the surrounding amino acid residues are more akin to metazoan adenylyl cyclases (see Fig. 1 B for an abbreviated sequence alignment). The expressed membrane-bound Paramecium guanylyl cyclase domain uses MgGTP as a substrate; it accepts MnATP as a substrate only slightly and is inactive with MgATP (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). Following successful strategies by others to produce soluble heterodimeric adenylyl cyclases (7.Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 8.Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (117) Google Scholar, 9.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google Scholar, 10.Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Crossref PubMed Scopus (109) Google Scholar, 11.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12.Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. 1997; 94: 2915-2920Crossref PubMed Scopus (64) Google Scholar, 13.Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (111) Google Scholar), we wished to assemble a protozoan heterodimeric guanylyl cyclase by expression inEscherichia coli of the catalytic domains either separately or covalently connected by a peptide linker. Here, we report on the expression and activity of such constructs. We then changed the purine nucleotide substrate specificity in this ciliate guanylyl cyclase with mammalian adenylyl cyclase topology by site-directed mutagenesis of two amino acids in the C1-positioned cytosolic loop. The inversion of the cytosolic loops was definitively proven by the successful generation of active adenylyl cyclase chimeras, which consisted only of cytosolic C2 cyclase domains, i.e. the mammalian adenylyl cyclase IIC2 or IXC2 and the C2-positioned domain of the Paramecium guanylyl cyclase. Radiochemicals were purchased from ICN or Amersham-Pharmacia Biotech; pQE vectors and nickel-NTA1 resin were from Qiagen. All enzymes were from Roche Molecular Biochemicals and New England Biolabs. To obtain the soluble constructs of theParamecium guanylyl cyclase cytosolic loops, we used a clone as a template in which all TAA/TAG-Gln codons had been mutated to the universal CAA/CAG-Gln triplets (GenBankTM accession numberAJ238859; Ref. 4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). For cloning purposes the C1a- (ParaGC-C1a) and C2- (ParaGC-C2) positioned cytosolic domains were fitted withSalI, MluI, and AscI restriction sites. The exact segment boundaries including the N-terminal His6-sequence and N- and C-terminal stuffer amino acids added to accommodate respective restriction sites are depicted in Fig.2 A. Conventional experimental protocols in molecular biology were followed to obtain the DNA constructs. A list of oligonucleotide sense and antisense primers and of synthetic mutagenic primers that were used in this study is available on request. The peptide linker AAGGPPAAGG in the construct ParaGC-C1aLParaGC-C2 was generated with two complementary oligonucleotide primers. The correctness of all constructs was verified by double-stranded DNA-sequencing. The constructs were cloned into a pQE30 expression vector that was modified to contain only a 5′-SalI and a 3′-AscI/HindIII cloning site. The C2 clone from rat type II adenylyl cyclase (GenBankTMaccession number M80550; IIC2) and a mouse type IX adenylyl cyclase clone (GenBankTM accession number Z50190) were kindly provided by Dr. C. Kleuss (Berlin) and Dr. F. Antoni (Glasgow), respectively. The C2 regions were cloned the with respective as and specific with and restriction sites for cloning of the into the pQE30 expression vector (see Fig. mutagenesis and was by the of and 1992; PubMed Scopus Google the site-directed mutagenesis and by to the C1a- and C2-positioned loops of the cyclase cloned into II as The constructs in the pQE30 expression were into the were in of containing and and with of were and in For C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google were in of containing a of inhibitors from were and were and were and the was for was by The were in and of was the resin was into a and with A with A with and were with of C containing The of the recombinant proteins with to adenylyl and guanylyl cyclase was Using we were to the protozoan constructs active upon on or by The of activity affected all protozoan constructs and not from to and on adenylyl or guanylyl cyclase were and all were from the affinity of activity was not to the proteins as analyzed by but to The proteins were analyzed by a mouse or a mouse Molecular were for in a of either or and 1 of were used as an of recombinant and The recombinant cyclase domains were on for to the by of and were and chromatography S. Schultz J.E. J. PubMed Scopus Google Scholar, Y. C. M. PubMed Scopus Google Scholar). The membrane-bound guanylyl cyclase from the Paramecium of two domains 1 A). The 155-kDa N-terminal domain resembles a P-type ATPase, and the C-terminal for a guanylyl cyclase of kDa with a structure of canonical mammalian adenylyl cyclases yet with reversed cytosolic C1a- and C2-positioned loops (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). For soluble constructs of cytosolic adenylyl cyclase loops have been (7.Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 8.Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (117) Google Scholar, 9.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google Scholar, 10.Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Crossref PubMed Scopus (109) Google Scholar, 11.Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12.Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. 1997; 94: 2915-2920Crossref PubMed Scopus (64) Google Scholar, 13.Tang W.-J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Crossref PubMed Scopus (111) Google Scholar, W.-J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, R.K. Dessauer C.W. R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar, C.W. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We wished to a soluble protozoan guanylyl cyclase from and C2-positioned loops and expressed the recombinant proteins in coli either or linked by a peptide in to W.-J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, R.K. Dessauer C.W. R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar, C.W. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We used a construct with an molecular of kDa i.e. amino acid in of the which to a slightly for in C2 of rat type II adenylyl cyclase. The protozoan C2-positioned loop with an molecular of kDa consisted of the of the transmembrane A). of the recombinant a the as the proteins and only a of a was present in Guanylyl cyclase activity of catalytic loops was not whereas a of the C1a- and C2-positioned loops had activity either or MgGTP as substrate the protozoan guanylyl cyclase loops had a affinity for each other to a active This is in to with the catalytic domains from mammalian adenylyl cyclases of soluble adenylyl cyclase activity mixing of cytosolic domains from and the of C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Crossref PubMed Scopus (86) Google and guanylyl cyclase of type and mutated constructs of Paramecium were with of the respective or and of each recombinant cytosolic domain or of the linked all affinity in were and in the C1-positioned domain and in the C2-positioned domain of the Paramecium guanylyl cyclase. not The of the was Data are from with similar The were of the C2-positioned in a were with of the respective or and of each recombinant cytosolic domain or of the linked all affinity in were and in the C1-positioned domain and in the C2-positioned domain of the Paramecium guanylyl cyclase. not The of the was Data are from with similar The were of the C2-positioned It was demonstrated that the activity of soluble constructs of mammalian adenylyl cyclases is or the catalytic loops are linked by a peptide (7.Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar). we linked the protozoan cytosolic domains a the order of the domains present in the reversed compared with mammalian adenylyl cyclases Fig. A). affinity the was by as a guanylyl cyclase activity was it not that in a of the expressed loops The of the of for MgGTP and were and and not from the with the linked construct and and to for the membrane-bound guanylyl cyclase domain expressed in or for the (4.Linder J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar, S. Schultz J.E. J. PubMed Scopus Google Scholar). of substrate were from to MgGTP and not from a substrate GTP The was to with the In with GTP as a substrate a of ATP guanylyl cyclase a specificity for GTP as a The of was A of was from the of an To the substrate specificity of the soluble guanylyl cyclase, we all constructs for adenylyl cyclase activity Using MgATP as a substrate, adenylyl cyclase activity was or of the respective guanylyl cyclase activity MnATP adenylyl cyclase activity was about of guanylyl cyclase and was similar to that with GTP as a substrate The crystal structure of a rat adenylyl cyclase type IIC2 and of a and rat to and and two or amino acids in the cyclases as responsible for purine nucleotide specificity (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, 6.Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google W.-J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). amino acids are all mammalian adenylyl and guanylyl cyclases and are In adenylyl cyclases Lys938 and the IIC2 catalytic domain with the and of the J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar, Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). In guanylyl cyclases the corresponding amino acids are and to the of rat soluble guanylyl which with the and the of the guanine of amino acid residues the respective substrate of mammalian guanylyl and adenylyl cyclases R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). to the Paramecium guanylyl cyclase, the C1-positioned domain is the of the catalytic of soluble guanylyl cyclases. Glu1681 in the C1-positioned loop is to and Ser1748 is to 1 In the a that is in mammalian guanylyl cyclases 1 This not the specific of GTP in the a the of the guanine and the This was a mutation the guanylyl cyclase activity to the of the A. C. J. Schultz G. Biochemistry. 1997; PubMed Scopus Google Scholar). To the of Glu1681 and Ser1748 in theParamecium domain in substrate we mutated to the adenylyl cyclase specific residues Lys and In we generated the the was not The constructs were enriched by affinity chromatography and by All recombinant proteins the molecular and were more or of Ser1748 to all cyclase of ATP or GTP as with as a guanylyl cyclase activity was by more and adenylyl cyclase activity was by more the of the adenylyl and guanylyl cyclase was only which that substrate was The then that the with the amino acid corresponding to to the for ATP or GTP substrate and to substrate The was in a purine nucleotide cyclase. in of two amino acids in mammalian membrane-bound or soluble guanylyl cyclases and in the mammalian had all of cyclase activity R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the guanylyl cyclase the nucleotide specificity was switched from GTP to of guanylyl cyclase were of type whereas adenylyl cyclase activity to and This demonstrated the inversion of the of the cytosolic loops in the ciliate guanylyl cyclase the amino acids were in the domain, whereas in mammalian adenylyl cyclases are in the C2 domain R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it demonstrated that which a that is in all mammalian guanylyl cyclases, is and a to had the In it the that the Paramecium guanylyl cyclase with a mammalian adenylyl cyclase membrane topology is a guanylyl cyclase we substrate specificity by site-directed mutagenesis of the protozoan domain as by the available of the mammalian adenylyl cyclase C2 (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, 6.Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). A novel for a cyclase with a canonical mammalian adenylyl cyclase topology was the in purine nucleotide This has been only for mammalian guanylyl cyclases R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The to the substrate specificity of a soluble mammalian adenylyl cyclase in a purine nucleotide cyclase R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the C1 domain of adenylyl cyclases type adenylyl was as a of for substrate by and by (5.Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (326) Google Scholar, R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hurley J.H. Hurley J.B. Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In adenylyl cyclases this is of a In soluble guanylyl cyclases the corresponding is an which to in the Paramecium C2-positioned loop. we mutated is in guanylyl in the ciliate C2-positioned domain to the adenylyl cyclase B and The construct was expressed in to a protein on a of the type with the guanylyl cyclase activity to and with MgGTP and the is adenylyl this have been it was to that also the adenylyl cyclase activity was to and with MgATP and MnATP as the of guanylyl to adenylyl cyclase that the of the C1-positioned loop is not all in substrate We that the was inactive which the The in which all residues and specific for were not cAMP formation that with the are in with substrate of the adenylyl cyclase chimera in which the Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). we the that a of the to substrate in other cyclase (see In theParamecium guanylyl cyclase the amino acid residues for substrate to for guanylyl cyclase adenylyl cyclase cyclase activity were with of the respective or Guanylyl cyclase activity was not with either All the domain were residues are in (see for in a were with of the respective or Guanylyl cyclase activity was not with either All the domain were residues are in (see for In the protozoan the molecular of the cytosolic and C2-positioned loops is reversed (see and J.U. Engel P. Reimer A. Krüger T. Plattner H. Schultz A. Schultz J.E. EMBO J. 1999; 18: 4222-4232Crossref PubMed Scopus (80) Google Scholar). To the of on we the of the C2-positioned domain for of cyclase activity with the catalytic C2 loop from mammalian adenylyl cyclases of rat type II and mouse type IX Fig. expression and IIC2 as a on a whereas the of IXC2 by of guanylyl cyclase activity was in protozoan C2 chimeras, whereas adenylyl cyclase were in with either IIC2 or IXC2 The were with MnATP as a substrate as compared with MgATP This was in the which was more active with MnATP the inversion of the cytosolic loops in the Paramecium guanylyl cyclase has been on an the C2-positioned domain from the ciliate guanylyl cyclase for a mammalian adenylyl cyclase domain. of the sequence theParamecium C2-positioned and mammalian domains, the which are to a were in the ciliate C2 loop. we used the for adenylyl cyclase with IIC2 or Whereas adenylyl cyclase activity was the for MnATP as a substrate was This that the to the of the C1 and C2 domains in adenylyl cyclases, not the domain not an active cyclase with mammalian domains not All membrane-bound mammalian adenylyl cyclases by the of and the forskolin, and by P site of M. Google Scholar). we the of canonical adenylyl cyclase or affected of the guanylyl cyclase constructs. This was not of the Paramecium guanylyl cyclase sequence that amino acid residues to for cyclase by and for such as (adenylyl cyclase numbering) are J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (680) Google Scholar). all were also by and In respective an of a was not The are of a chimera of and the of a soluble rat guanylyl cyclase, which also was to the adenylyl cyclase S. Kleuss C. Biochemistry. 1999; PubMed Scopus Google Scholar). adenylyl cyclase by and to constructs the mammalian C1 and C2 P site inhibitors the of the cyclases with C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). is M. Google Scholar). The available P site for adenylyl cyclases is with of 1 for the and in the upon by In a similar guanylyl cyclase is by R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We the of and on the constructs generated In P site soluble Paramecium guanylyl cyclase constructs was 1 not guanylyl cyclase and 1 by about guanylyl cyclase was with as a i.e. in a activity guanylyl cyclase activity was with as an i.e. in a activity was more i.e. was and specific as as the purine was The of the by the in the of a soluble guanylyl cyclase had on the of we the adenylyl cyclase reconstituted from the mutated Paramecium domains and and from the chimeras, i.e. and 1 adenylyl cyclase of all reconstituted enzymes by about was 1 i.e. we that substrate and P site specificity to in constructs as reported for mammalian constructs R.K. A. J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the of the P site inhibitors compared with mammalian cyclases. The then of this was the The protein that is in for the of the group of the nucleotide in with the of two Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). The amino acids are of the C1 and in the domain and and IIC2 Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). from respective sequence all of amino acid residues are present in the corresponding C2- and C1-positioned domains of the protozoan guanylyl cyclase. are of the C2-positioned and in the C2-positioned loop and and in the catalytic C1-positioned loop (see the abbreviated in we that the amino acid for the site is present in the protozoan guanylyl cyclase and that the of of P site inhibitors has a In mammalian adenylyl cyclases loops a substrate ATP to a specific and active site and the heterotrimeric proteins either or such a which the substrate group and the catalytic residues Sunahara R.K. G. Gilman A.G. Sprang S.R. Science. 1999; PubMed Scopus Google Scholar). all of the amino acid residues for such a are present in theParamecium guanylyl cyclase, we that it in a similar to that of a mammalian adenylyl cyclase. it is that it also an to the into a activity which then affected by P site inhibitors mammalian adenylyl cyclases have to for the of of the heterotrimeric protein in Paramecium U. T. U. and J. In in the of a and a protein that the substrate, have been we have to for the of a protozoan protein that the Ca2+ in the guanylyl cyclase S. Schultz J.E. J. PubMed Scopus Google Scholar). In this the of the 155-kDa N-terminal P-type domain the more The of a P-type with a guanylyl cyclase as a mammalian adenylyl cyclase in a sequence to novel as M. Science. 1999; PubMed Scopus Google of cyclase by with 1 cyclase cyclase and was used in all residues are in (see for in a and was used in all residues are in (see for We C. Kleuss and (Berlin) for a clone of the adenylyl cyclase type IIC2 and a of and Dr. F. for a clone of the mouse type IX adenylyl cyclase.

Gregory A. Poland, Robert M. Jacobson

No abstract is available for this record.

Tatsuaki Okamoto

No abstract is available for this record.

Christian Cachin, Jan Camenisch

No abstract is available for this record.

Guillaume Poupard, Jacques Stern

No abstract is available for this record.

Ronald Cramer, Ivan Damgård, Philip MacKenzie

No abstract is available for this record.

Steen Olsen

Diabetic nephropathy is one of the most frequent and clinically important complications to diabetes mellitus (DM), affecting approximately 40% of patients who have had diabetes for more than 20 years and contributing to a substantial part of patients entering into end-stage renal failure programmes. Our knowledge of the clinical development and morphological basis for diabetic renal disease has augmented tremendously during the last half century [1,2] and during the last decade we have been able to understand at least some elements of its pathogenesis. Nephropathy in a patient suffering from DM may, however, not be related directly to the diabetic disease but may be a complicating renal disease, not due to the diabetic metabolic abnormality. The situation is further complicated since logically both lesions may be present in the same patient. While it was formerly held that non-diabetic renal disease was of minor importance in DM, this seems not so obvious to-day, and opinions of investigators about prevalence of non-diabetic renal disease in DM, as revealed by renal biopsies, have been conflicting. The correct diagnosis is important for the patient since prognosis and treatment may vary according to the underlying cause. Furthermore: if a substantial part of renal disease in diabetics had other causes than the diabetic metabolic derangement, this must be a confounding factor in the interpretation of intervention trials. The classical diabetic nephropathy is morphologically primarily a glomerulopathy, characterized clinically by proteinuria and progressive decrease of glomerular filtration. Tubular atrophy and interstitial fibrosis is a common final pathway for many renal diseases including diabetic nephropathy. These lesions have formerly been interpreted as ischaemic sequels to vascular and glomerular lesions but they may also occur in diabetics with only mild vascular and glomerular lesions and at an early stage of diabetic renal disease. In some reports this was even rather frequent [3,4] and it is today not at all obvious that tubulointerstitial lesions are of ischaemic origin. A competing hypothesis relates them to the diabetic state, just as glomerulopathy [5–7]. This problem will not be addressed here, I have been asked to provide a brief presentation of problems related to the differentiation of non-diabetic from diabetic glomerular lesions in renal biopsies. Since our problem is one of differential diagnosis it is first necessary to give a brief review of glomerular lesions, which can be regarded with confidence as being directly caused by the diabetic metabolic abnormalities. Following the first seminal report by Kimmelstiel and Wilson [8] and later on worked out in great detail by others, we have now a clear idea of the histopathology of diabetic renal disease and its progress with time. We know now, that at the onset of insulin dependent DM (IDDM) (in this type the zero time is well defined) there is enlargement of the glomeruli indicating increased glomerular function, but no other structural lesions by light microscopy (LM) or electron microscopy (EM). The first ultrastructural lesions (widening of the mesangial matrix and thickening of the peripheral glomerular basement membrane) can be morphometrically identified 2–3 years after onset [9]. Changes can usually not be seen by LM until several years of diabetic disease. They are described in two forms. In the diffuse form there is a widening of all mesangial regions as well as thickening of the peripheral capillary basement membranes, corresponding to what is already apparent at an earlier time from morphometry on EM pictures. The diffuse lesion is most conspicuous in PAS-stained sections. In the nodular type some of the mesangial regions take on the appearance of round nodules which are acellular in their centres but surrounded by a rim of mesangial nuclei. While these types are sometimes described as separate histologic entities, there are good reasons to regard the nodular type as a further development of the diffuse type. Missing a formal proof of this (e.g. by the study of sequential biopsies from the same patients), it is clear that nodules are formed from mesangial matrix, that they occur later in the disease than the diffuse lesion and that nodules never occur without associated marked diffuse lesions. To this account of what is regarded as the most important glomerular lesions may be added that hyaline (exudative) lesions sometimes occur in the form of capsular drops and hyaline caps. It is unclear whether mesangial hypercellularity is a feature of glomerular diabetic lesions. Using quantitative technique, Wehner and Anders [10] as well as Kimmelstiels group [11] found a significant increase of mesangial nuclei in nodular and diffuse diabetic glomerulopathy. On the other hand, Østerby, counting on EM montages, did not find hypercellularity in the early stage of juvenile DM (0–5 years) [12]. In experimental diabetic nephropathy there is early mesangial proliferation and increased expression of growth factors [13]. Using immunofluorescence, a diffuse, linear localization of IgG in glomerular capillary walls, and sometimes also of small quantities of other plasma components, is frequent. There is general agreement that this is due to non-specific trapping of plasma components along the glomerular capillary wall, and they are not associated with presence of deposits on EM. In parallel with the progress of glomerulopathy, arteriolar sclerosis and hyalinosis develop. The hyaline arteriolosclerosis affects the afferent as well as the efferent arteriole, which is not the case with non-diabetic arteriolosclerosis. Otherwise there are no differences between the structure of arteriosclerosis and arteriolosclerosis in diabetics and non-diabetics. Arteriosclerosis occur earlier and in more severe degree in diabetics than in the non-diabetic population. It is therefore not unexpected that arteriosclerosis and focal scars are frequent in biopsies from patients with DM, especially non-insulin dependent DM (NIDDM) which occurs in older patients. It is generally held that GN complicating IDDM is comparatively rare, probably around 2–3% in unselected cases with proteinuria and duration of diabetes of more than 10 years. Some reports have shown an impressively high prevalence of GN in NIDDM. The widely diverging results of the reports indicate, however, that something must have gone wrong. We have recently analysed 10 renal biopsy studies (including our own) specifically dealing with NIDDM [3,4,14–21]. The rates of GN in these series vary between 0 and 66% and other complicating renal diseases were reported to be present in between 0 and 20%. These diverging results can of course be due to selection criteria (only one was a population based cross-sectional study [15]) or geographical differences, but the criteria for diagnosis of GN and interobserver variation in the pathologists interpretation of structural lesions may also be responsible. As most types of glomerulonephritis are due to deposition of antibodies in the glomerular capillary walls or the mesangium, immunohistochemical investigation will often, together with the LM characteristics of the glomerular lesions, provide an unambigous diagnosis. This applies to the most common GN form, IgA nephropathy, as well as several other such as membranous GN, membranoproliferative GN, postinfectious, complex mediated GN, and GN associated with systemic lupus erythematosus. GN in systemic vasculitis may be without immune deposits, but the LM picture (crescentic or focal, segmental proliferative GN) is easily distinguished from the lesions seen in diabetes. While the identification of these types in the reports of complicating renal disease in NIDDM must be regarded as unproblematic, this is certainly not the case for two other types: mesangial proliferative GN and minimal change disease. There is therefore reason to discuss these entities specifically. This is a descriptive term indicating diseases with increased mesangial cell number (more than three mesangial cells per mesangial region in at least 80% of all glomeruli). This light microscopical picture (as defined by a WHO committee [22]) can be seen in several types of GN such as IgA nephropathy, in the resolution phase of postinfectious GN, in some cases of GN associated with SLE etc. These types have all heavy immune deposits and their composition (immunoglobulin classes) pattern and location makes them easy to distinguish from diabetic glomerulopathy. There is, however, a comparatively rare mesangioproliferative GN without immune deposits. We meet two problems with this type if it is detected in a patient with diabetes. One is that mesangial hypercellularity (as discussed above) may be a feature of diabetic glomerulopathy, at least in some stages of its development. The other is that the usual evaluation of mesangial cell number is subjective, and as such dubious, especially in cases with only weak or moderate increase of the number of cells. A biopsy diagnosis in a diabetic patient of slight or moderate mesangioproliferative GN without immune deposits must therefore be regarded as questionable. This term indicates, according to the WHO classification of glomerular disease [22], conditions with normal glomerular structure or minor glomerular abnormalities such as slight mesangial hypercellularity. Clinically it comprises minimal change nephrotic syndrome as well as mild or moderate persistent isolated proteinuria. Significant immune deposits detected by immune histochemistry or on EM are not present. Podocyte foot processes are more or less effaced, but this is unspecific and can be seen in all renal diseases with proteinuria [23]. The pathogenesis is unknown. The application of this diagnosis in a patient with DM presents serious problems. Normal glomeruli by LM and no deposits by immunofluorescence or electron microscopy is completely compatible with early diabetic glomerulopathy with slight or even moderate albuminuria. On the other hand, patients suffering from DM may of course also be affected by other glomerular diseases, possibly even with a higher incidence that the non-diabetic population. Among these complicating conditions are the `classical', well defined glomerular immune disorders mentioned above, but also the less well characterized renal diseases collected under the term minimal change nephropathy. To this may be added that it is well known that a collection of biopsies with normal glomeruli may comprise cases of the more serious renal disease, focal, segmental glomerulosclerosis and hyalinosis, due to missing representation of the abnormal glomeruli in a biopsy with a restricted number of glomeruli. The correct diagnosis may in such cases be made in a later biopsy from the patient. It will be clear from the above discussion that it is hazardous to make the diagnosis of glomerulonephritis based upon light microscopy alone, especially in the situation dealt with here. This may underestimate the prevalence of GN in DM [24], but probably also overestimate if slight or moderate mesangial hypercellularity alone is used as a criterium. Immunofluorescence microscopy should always be added to the investigation of such cases, and optimally also EM. The clinical context is of course also significant. If it is strongly different from diabetic nephropathy, for example, if nephrotic syndrome appears suddenly early in the course of the diabetic disease, the diagnosis of minimal change nephrotic syndrome becomes convincing. Haematuria, which is not characteristic for diabetic nephropathy may indicate GN. In the ten reports discussed above, many other renal diseases than GN was found, among these amyloidosis and myeloma which can also involve glomeruli. In amyloidosis there is deposition of the abnormal protein in the mesangial matrix and along the capillary walls. The morphology is usually different from diffuse and nodular diabetic glomerulopathy, but nodules may nevertheless be seen. Mesangial nodules can also be seen in myeloma and related dysproteinaemias [25]. The conclusion from these considerations must be the following. If a patient suffering from DM develops clinical nephropathy (proteinuria) there may logically be three reasons. Diabetic glomerulopathy may be in progress, non-diabetic renal disease alone may be responsible for the nephropathy, or both may be present. Non-diabetic nephropathy is most often glomerulonephritis, but also other renal diseases, not related to the diabetic disease (amyloidosis etc). A renal biopsy studied by light microscopy and immune fluoroscence may provide the key to correct diagnosis, either presenting characteristic diabetic glomerulopathy alone or unequivocal GN with glomerular immune deposits. There are, however, situations where a biopsy cannot lead to a reliable diagnosis. Renal diseases associated with normal glomerular structure (minimal change nephropathy) or with slight or moderate alterations which cannot with certainty be differentiated from diabetic renal disease in early stages (mesangial proliferative GN without immune deposits) may of course also occur in diabetics and proteinuria may be related to one of these diseases. Such a patient may belong to the category which never develop diabetic renal disease or he or she may have diabetic glomerulopathy at an early stage where there is proteinuria but no structural changes on LM. Even if the biopsy is investigated with EM the situation is not necessarily unambigous: slight or moderate thickening of the glomerular basement membrane or mesangial matrix may be present in patients with longstanding diabetes without proteinuria. We have expressed the opinion that complicating GN in NIDDM is present in about 10% [14], other investigators have reported 25% [15] or even more. Actually it may be impossible at the moment to gain reliable knowledge of the prevalence due to the problems discussed above. The group of patients with DM and normal glomeruli or slight or moderate mesangial proliferation should be investigated carefully in order to solve this important problem.

Mike Burmester, Yvo Desmedt, Toshiya Itoh, Kouichi Sakurai · 5 authors

No abstract is available for this record.

Birgit Pfitzmann, Ahmad‐Reza Sadeghi

No abstract is available for this record.

J. Mohajeri

No abstract is available for this record.

Markus Jakobsson, Claus-Peter Schnorr

We study the notion of meta-proofs, which, as the name indicates, are proofs about proofs. We employ the notion of meta-proofs to produce a highly efficient oblivous proof of correct exponentiation. It is minimum-knowledge independently of whether the input is valid or not, a property that does not hold for many other protocols (that are zero-knowledge only for valid inputs.) This has direct security implications to multiparty protocols, where the protocols we demonstrate — one interactive and one non-interactive — can be employed to obtain protocol robustness at a low cost. As a result of potential independent interest, we show how to turn any standard discrete log signature scheme into a scheme for proving equality of discrete logarithms. We demonstrate our method using the Schnorr signature scheme.

Oded Goldreich, Amit Sahai, Salil Vadhan

No abstract is available for this record.

Giovanni Di Crescenzo, Rafail Ostrovsky

No abstract is available for this record.

Jan Camenisch, Markus Michels

&lt;p&gt;This paper presents the first efficient statistical zero-knowledge protocols to prove statements such as:&lt;br /&gt;A committed number is a pseudo-prime.&lt;br /&gt;A committed (or revealed) number is the product of two safe primes, i.e., primes p and q such that (p - 1)=2 and (q - 1)=2 are primes as well.&lt;br /&gt;A given value is of large order modulo a composite number that consists of two safe prime factors.&lt;/p&gt;&lt;p&gt;So far, no methods other than inefficient circuit-based proofs are known for proving such properties. Proving the second property is for instance necessary in many recent cryptographic schemes that rely on both the hardness of computing discrete logarithms and of difficulty computing roots modulo a composite.&lt;br /&gt;The main building blocks of our protocols are statistical zero-knowledge proofs that are of independent interest. Mainly, we show how to prove the correct computation of a modular addition, a modular multiplication, or a modular exponentiation, where all values including the modulus are committed but not&lt;br /&gt;publicly known. Apart from the validity of the computation, no other information about the modulus (e.g., a generator which order equals the modulus) or any other operand is given. Our technique can be generalized to prove in zeroknowledge&lt;br /&gt;that any multivariate polynomial equation modulo a certain modulus is satisfied, where only commitments to the variables of the polynomial and a commitment to the modulus must be known. This improves previous results,&lt;br /&gt;where the modulus is publicly known.&lt;br /&gt;We show how a prover can use these building blocks to convince a verifier that a committed number is prime. This finally leads to efficient protocols for proving that a committed (or revealed) number is the product of two safe primes. As a consequence, it can be shown that a given value is of large order modulo a&lt;br /&gt;given number that is a product of two safe primes.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;&lt;p&gt;Keywords. RSA-based protocols, zero-knowledge proofs of knowledge, primality tests.&lt;/p&gt;

Oded Goldreich, Shafi Goldwasser

We show simple constant-round interactive proof systems for problems capturing the approximability, to within a factor of n , of optimization problems in integer lattices, specifically, the closest vector problem (CVP) and the shortest vector problem (SVP). These interactive proofs are for the coNP direction; that is, we give an interactive protocol showing that a vector is far from the lattice (for CVP) and an interactive protocol showing that the shortest-lattice-vector is long (for SVP). Furthermore, these interactive proof systems are honest-verifier perfect zero-knowledge. We conclude that approximating CVP (resp., SVP) within a factor of n is in N P ∩co A M . Thus, it seems unlikely that approximating these problems to within a n factor is NP-hard. Previously, for the CVP (resp., SVP) problem, Lagarias et al. (1990, Combinatorica 10 , 333–348), Håstad (1988, Combinatorica 8 , 75–81), and Banaszczyk (1993, Math. Annal. 296 , 625–635) showed that the gap problem corresponding to approximating CVP (resp., SVP) within n is in N P ∩co N P . On the other hand, Arora et al. (1997, J. Comput. System Sci. 54 , 317–331) showed that the gap problem corresponding to approximating CVP within 2 log 0.999 n is quasi-NP-hard.

Donald Beaver

No abstract is available for this record.

Liqun Chen

No abstract is available for this record.