Pig Insulin Research Articles

Functional expression of recombinant insulins in Saccharomyces cerevisiae

BackgroundSince 1982, recombinant insulin has been used as a substitute for pancreatic insulin from animals. However, increasing demand in medical and food industries warrants the development of more efficient production methods. In this study, we aimed to develop a novel and efficient method for insulin production using a yeast secretion system.MethodsHere, insulin C-peptide was replaced with a hydrophilic fusion partner (HL18) containing an affinity tag for the hypersecretion and easy purification of proinsulin. The HL18 fusion partner was then removed by in vitro processing with the Kex2 endoprotease (Kex2p), and authentic insulin was recovered via affinity chromatography. To improve the insulin functions, molecular chaperones of the host strain were reinforced via the constitutive expression of HAC1.ResultsThe developed method was successfully applied for the expression of cow, pig, and chicken insulins in yeast. Moreover, biological activity of recombinant insulins was confirmed by growth stimulation of cell line.ConclusionsTherefore, replacement of the C-peptide of insulin with the HL18 fusion partner and use of Kex2p for in vitro processing of proinsulin guarantees the economic production of animal insulins in yeast.

Hazardous Shortcuts in Standard Binding Free Energy Calculations.

Calculating the standard binding free energies of protein-protein and protein-ligand complexes from atomistic molecular dynamics simulations in explicit solvent is a problem of central importance in computational biophysics. A rigorous strategy for carrying out such calculations is the so-called "geometrical route". In this method, two molecular objects are progressively separated from one another in the presence of orientational and conformational restraints serving to control the change in configurational entropy that accompanies the dissociation process, thereby allowing the computations to converge within simulations of affordable length. Although the geometrical route provides a rigorous theoretical framework, a tantalizing computational shortcut consists of simply leaving out such orientational and conformational degrees of freedom during the separation process. Here the accuracy and convergence of the two approaches are critically compared in the case of two protein-ligand complexes (Abl kinase-SH3:p41 and MDM2-p53:NVP-CGM097) and three protein-protein complexes (pig insulin dimer, SARS-CoV-2 spike RBD:ACE2, and CheA kinase-P2:CheY). The results of the simulations that strictly follow the geometrical route match the experimental standard binding free energies within chemical accuracy. In contrast, simulations bereft of geometrical restraints converge more poorly, yielding inconsistent results that are at variance with the experimental measurements. Furthermore, the orientational and positional time correlation functions of the protein in the unrestrained simulations decay over several microseconds, a time scale that is far longer than the typical simulation times of the geometrical route, which explains why those simulations fail to sample the relevant degrees of freedom during the separation process of the complexes.

Extracellular hemoglobin combined with an O2 -generating material overcomes O2 limitation in the bioartificial pancreas.

The bioartificial pancreas encapsulating pancreatic islets in immunoprotective hydrogel is a promising therapy for Type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O2 supply after transplantation remains a major challenge. In this study, we address the O2 limitation by combining silicone-encapsulated CaO2 (silicone-CaO2 ) to generate O2 with an extracellular hemoglobin O2 -carrier coencapsulated with islets. We showed that the hemoglobin improved by 37% the O2 -diffusivity through an alginate hydrogel and displayed antioxidant properties neutralizing deleterious reactive O2 species produced by silicone-CaO2 . While the hemoglobin alone failed to maintain alginate macroencapsulated neonate pig islets under hypoxia, silicone-CaO2 alone or combined to the hemoglobin restored islet viability and insulin secretion and prevented proinflammatory metabolism (PTGS2 expression). Interestingly, the combination took the advantages of the two individual strategies, improved neonate pig islet viability and insulin secretion in normoxia, and VEGF secretion and PDK1 normalization in hypoxia. Moreover, we confirmed the specific benefits of the combination compared to silicone-CaO2 alone on murine pseudo-islet viability in normoxia and hypoxia. For the first time, our results show the interest of combining an O2 provider with hemoglobin as an effective strategy to overcome O2 limitations in tissue engineering.

Acute effects of zinc and insulin on arcuate anorexigenic proopiomelanocortin neurons.

Acute insulin administration hyperpolarized, with concomitant decrease of firing rate, a subpopulation of arcuate proopiomelanocortin (POMC) and neuropeptide Y/agouti-related peptide cells. This rapid effect on cellular activity has been proposed as a cellular correlate of insulin effects on energy balance and glucose homoeostasis. Recent evidence suggests that zinc in mammalian insulin formulations is required for the insulin-induced inhibition of arcuate POMC neurons, while guinea pig insulin, which fails to bind zinc, activates POMC neurons in mice. Here, we tested the effects of zinc and insulin formations on arcuate POMC neurons. Effects of zinc and insulin formulations were assessed through whole-cell patch clamp recordings on transgenic mice in vitro. Insulin formulations containing zinc hyperpolarized POMC neurons. Zinc also hyperpolarized arcuate POMC neurons, albeit at much higher concentration than found in various insulin formulations. Chelation of zinc inhibited the zinc-induced hyperpolarization of POMC neurons, whereas effects of insulin on POMC cellular activity were unchanged after chelation. Zinc-free insulin also hyperpolarized arcuate POMC neurons. Insulin failed to hyperpolarize POMC neurons deficient for insulin receptors, suggesting that insulin receptors are required for these effects. Activation of POMC neurons by guinea pig insulin was independent of insulin receptors but was inhibited by PDGF receptor antagonism or loss of TRPC5 channel subunits. Together, these findings suggest that insulin inhibited arcuate POMC neurons independent of zinc and highlights a possible role of putative PDGF receptors in the acute effects of guinea pig insulin.

Co-expression of mFat-1 and pig IGF-1 genes by recombinant plasmids in modified chitosan nanoparticles and its synergistic effect on mouse immunity

To develop a cost-effective molecular regulator to improve growth metabolism and immunity of animals, a recombinant plasmid co-expressing fatty acid desaturase (mFat-1) and pig insulin growth like factor 1 (IGF-1) genes was constructed by the 2 A self-cleavage technique. After entrapment within modified chitosan nanoparticles (chitosan modified with polyethyleneglycol–polyethylenimine, CPP), the recombinant plasmid was injected intramuscularly into mice. Compared with controls, co-expression of mFat-1 and IGF-1 significantly raised the level of serum IGF-1, and increased the liver and muscle docosa hexaenoic acid (DHA) content. Th and Tc cell levels were also elevated, as were expression levels of serum IL-4 and IL-6 genes. These results demonstrate that the immunity and metabolism of an animal can be effectively improved by co-expression of mFat-1 and IGF-1 genes in vivo, which may contribute to further development of novel immunomodulators with beneficial effects on growth metabolism and immunity.

Expression, receptor binding, and biophysical characterization of guinea pig insulin desB30: a monomeric insulin variant.

Here we report, for the first time, the heterologous expression of desB30 guinea pig insulin (GI desB30) in the yeast Saccharomyces cerevisiae. The affinities of GI desB30 for the insulin receptor A and the IGF-I receptor were also quantified for the first time. Small-angle X-ray scattering and analytical ultracentrifugation studies confirmed that GI desB30 did not form dimers or hexamers, in contrast to human insulin. Size-exclusion chromatography connected to inductively coupled plasma mass spectrometry revealed that GI desB30 has affinity towards several divalent metal ions. These studies did not indicate the formation of any larger structures of GI desB30 in the presence of various divalent metal ions, but did indicate that GI desB30 has an affinity towards Mn, Co, and Cu ions. Finally, the low affinity for the insulin receptor and the very low affinity for the IGF-I receptor by GI desB30 were quantified.

Pancreatic Transdifferentiation in Porcine Liver Following Lentiviral Delivery of Human Furin–Cleavable Insulin

Type I diabetes mellitus (TID) results from the autoimmune destruction of the insulin-producing pancreatic β-cells. Gene therapy is one strategy being actively explored to cure TID by affording non–β-cells the ability to secrete insulin in response to physiologic stimuli. In previous studies, we used a novel surgical technique to express furin-cleavable human insulin (INS-FUR) in the livers of streptozotocin (STZ)–diabetic Wistar rats and nonobese diabetic (NOD) mice with the use of the HMD lentiviral vector. Normoglycemia was observed for 500 and 150 days, respectively (experimental end points). Additionally, some endocrine transdifferentiation of the liver, with storage of insulin in granules, and expression of some β-cell transcription factors (eg, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4) and pancreatic hormones in both studies. The aim of this study was to determine if this novel approach could induce liver to pancreatic transdifferentiation to reverse diabetes in pancreatectomized Westran pigs. Nine pigs were used in the study, however only one pig maintained normal fasting blood glucose levels for the period from 10 to 44 days (experimental end point). This animal was given 2.8 × 109 transducing units/kg of the lentiviral vector expressing INS-FUR. A normal intravenous glucose tolerance test was achieved at 30 days. Reverse-transcription polymerase chain reaction analysis of the liver tissue revealed expression of several β-cell transcription factors, including the key factors, Pdx-1 and Neurod1, pancreatic hormones, glucagon, and somatostatin; however, endogenous pig insulin was not expressed. Triple immunofluorescence showed extensive insulin expression, as was previously observed in our studies with rodents. Additionally, a small amount of glucagon and somatostatin protein expression was seen. Collectively, these data indicate that pancreatic transdifferentiation of the liver tissue had occurred. Our data suggest that this regimen may ultimately be used clinically to cure TID, however more work is required to replicate the successful reversal of diabetes in increased numbers of pigs.

Fetal Pancreas as a Source for Islet Transplantation

The early hope that islet transplantation might offer an attractive treatment modality for late-stage type 1 diabetes has been tempered over the last decade, largely due to the observation that the transplants exhibit reduced insulin production after a few years (1). This limitation could be associated with some form of immune rejection or the inability of the implanted islets to replenish dying islets via stem cell differentiation and self-renewal. Studies over the past three decades indicate that both immunogenicity and poor self-renewal might potentially be addressed by the use of human fetal pancreatic precursor tissues (2,3). However, considering the ethical issues associated with the use of human fetal tissues and the proven efficacy of pig insulin in humans, the focus of such studies has shifted to xenotransplantation of pig embryonic pancreatic tissue. While several studies demonstrated the remarkable regenerative capacity of embryonic day (E)28–E42 pig pancreatic tissues in various rodent models (4–7) and the ability of E42 tissue to correct diabetes in nonhuman primates (NHP) (8), the issue of immunogenicity remained highly controversial. In particular, at the heart of this debate was the suggestion by Rogers et al. that pig embryonic pancreatic (6) or renal (9) tissues harvested at E28 can completely evade graft rejection in fully immune competent mice or rats and, …

Xenotransplantation of embryonic pig pancreas for treatment of diabetes mellitus in non-human primates

Transplantation therapy for diabetes in humans is limited by the low availability of human donor whole pancreas or islets. Outcomes are complicated by immunosuppressive drug toxicity. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce transplant immunogenicity. Pig insulin is biologically active in humans. In that regard the pig is an appropriate xenogeneic organ donor. Insulin-producing cells originating from embryonic pig pancreas obtained very early following pancreatic primordium formation [embryonic day 28 (E28)] engraft long-term in rhesus macaques. Endocrine cells originating from embryonic pig pancreas transplanted in host mesentery migrate to mesenteric lymph nodes, engraft, differentiate and improve glucose tolerance in rhesus macaques without the need for immune suppression. Transplantation of embryonic pig pancreas is a novel approach towards beta cell replacement therapy that could be applicable to humans.

Need for xenotransplantation in the treatment of type 1 diabetes

Type 1 diabetes (T1D) currently affects approximately 28 million patients with a rising incidence worldwide. Despite improvement of intensive insulin therapy, only a subgroup of patients reached near-normal blood glucose control. Prospective studies have shown that 30–40% of patients with T1D are at risk to develop devastating micro- or macrovascular complications. In the intensive insulin treatment arm of DCCT/EDIC study the cumulative incidences of proliferative retinopathy, nephropathy and cardiovascular diseases were 21%, 9% and 9% after 30 years of diabetes, respectively [1]. In addition, about 15% of patients with a disease history of 20–25 years have the problem of hypoglycemia unawareness due to lack of hormonal counter-regulation [2]. These serious complications lead to a significant reduction of life-expectancy as compared to non-diabetic individuals. Thus, there is an urgent need for more effective and durable management of diabetes. Transplantation of human beta cells using isolated islets or pancreas provided evidence that T1D can be cured by cell replacement therapies. Pancreas transplantation results in a significant reduction of vascular complication and reduced total mortality [3]. After islet cell transplantation 3-year insulin independency, preserved graft function (detectable C-peptide) and elimination of severe hypoglycemia have been reported in 23%, 52% and 70% by the collaborative islet transplant registry [4]. However, the total number of islet allografts (<100/years) and pancreas transplants (approximately 1800/year) worldwide is insufficient in relation to the number of potential recipients in whom islet/pancreas transplantation may be beneficial or even life-saving. In the last years there have been impressive advances in the field of beta cell generation from induced pluripotent stem cells/embryonic stem cells and xenograft transplantation. It has been shown that novel immunosuppressive regimen can induce long-lasting pig islet survival in nonhuman primates [5,6] and the use of fetal pig pancreas reduces immunogenicity [7]. Transgenic pigs expressing immunosuppressive and cytoprotective factors in beta cells have been generated. In addition, novel techniques of islet encapsulation in biochambers may allow long-term islet function and pathogen-free pig herds have been recently established to overcome the problem of zoonosis. The use of pig islets has additional advantages: (i) pig insulin is safe since it has been used for decades in the treatment of T1D; (ii) porcine pancreas is easily available on demand making it possible to reduce ischemia injury and pretreatment of recipients; (iii) pig islets do respond to glucose in a similar way as human islets. The last two points strongly favor pig islets as compared to neo-beta cells derived from stem cells, which were reported to be not fully matured and possess the risk of tumor formation (teratomas) [8,9]. The striking advances in pig islet transplantation suggest that it may be possible to overcome the major immunological barriers to pig islets in recipients so that clinical trials can be started in humans within the next years. Therefore, porcine islet xenografts may be the most realistic option to offer cell replacement therapy to a higher number of patients with T1D at risk for severe diabetes-associated complications. When this approach is successful the large-scale availability of pig islets would also open the window for other applications such as transplantation in insulin-treated patients with type 2 diabetes in whom insulin resistance currently excludes islet cells transplantation due to the demand of high insulin levels/high number of islets.

67 PIG OOCYTES WITH A LARGE PERIVITELLINE SPACE MATURED IN VITRO HAVE GREATER DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER

The size of perivitelline space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P &lt; 0.05. In Experiment 1, oocytes with a larger PVS had higher (P &lt; 0.05) levels of intracellular GSH (1.0 pixels/oocyte v. 0.6 pixels/oocyte) and blastocyst formation (54% v. 37%) after PA than oocytes with smaller PVS. In Experiment 2, maturation culture of oocytes in PZM-3 with reduced (61.6 mM) NaCl concentration significantly increased (P &lt; 0.05) the size of the PVS (5.2 μM v. 3.3 μM) compared with control oocytes that were matured in PZM-3 containing 108 mM NaCl, although the treatment did not alter the nuclear maturation. Moreover, oocytes with increased PVS expressed more CDK1, PCNA, and ERK2 mRNA and had higher (P &lt; 0.05) intracellular GSH levels (1.6 pixels/oocyte v. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52% v. 41%) and SCNT (32% v. 18%) compared with control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation resulting from the enhanced GSH level and transcription factor expression and that enlargement of PVS by the culture in low-NaCl medium also improves developmental competence of pig oocytes. This work was supported by grants (#20070301034040 and #20080401034072) from the BioGreen 21 Program (Rural Development Administration, Republic of Korea).

Pig Embryonic Pancreatic Tissue as a Source for Transplantation in Diabetes

OBJECTIVEDefining an optimal costimulatory blockade–based immune suppression protocol enabling engraftment and functional development of E42 pig embryonic pancreatic tissue in mice.RESEARCH DESIGN AND METHODSConsidering that anti-CD40L was found to be thrombotic in humans, we sought to test alternative costimulatory blockade agents already in clinical use, including CTLA4-Ig, anti-LFA1, and anti-CD48. These agents were tested in conjunction with T-cell debulking by anti-CD4 and anti-CD8 antibodies or with conventional immunosuppressive drugs. Engraftment and functional development of E42 pig pancreatic tissue was monitored by immunohistology and by measuring pig insulin blood levels.RESULTSFetal pig pancreatic tissue harvested at E42, or even as early as at E28, was fiercely rejected in C57BL/6 mice and in Lewis rats. A novel immune suppression comprising anti-LFA1, anti-CD48, and FTY720 afforded optimal growth and functional development. Cessation of treatment with anti-LFA1 and anti-CD48 at 3 months posttransplant did not lead to graft rejection, and graft maintenance could be achieved for >8 months with twice-weekly low-dose FTY720 treatment. These grafts exhibited normal morphology and were functional, as revealed by the high pig insulin blood levels in the transplanted mice and by the ability of the recipients to resist alloxan induced diabetes.CONCLUSIONSThis novel protocol, comprising agents that simulate those approved for clinical use, offer an attractive approach for embryonic xenogeneic transplantation. Further studies in nonhuman primates are warranted.

A Novel Role for Factor VIII and Other Coagulation Factors in Organ Size Control

Despite great progress in molecular genetics and embryogenesis, size control of tissues and organs remains a mystery. In general, it seems that extrinsic mechanisms are associated with nutrition or systemic growth factors signaling such as the Insulin/PI3K or the TOR pathways, while intrinsic mechanisms are likely linked to patterning morphogens and apoptosis signaling complexes. Very recently we defined optimal ‘windows’ of gestation of human and pig embryonic precursor tissues which afford optimal growth and development of liver, pancreas and spleen upon implantation into NOD-SCID mice. This type of study enables not only a rational selection of tissue for transplantation but, among many parameters, it also provides a system and ability to manipulate organ size control. Using this system we surprisingly found that pig embryonic implants grow to a significantly larger size in Factor VIII Knock Out (KO) NOD-SCID mice. The average size of E42 pig spleen implant grown for 3 months in Factor VIII KO SCID recipients was 6.78 ± 2.16gr compared to 2.46 ± 0.82gr in non-hemophilic NOD-SCID recipients (p<0.05). Histological examination of the growing spleen implants revealed normal growth, development and vascularization patterns comparable to those found in the corresponding Factor VIII wild type mice, ruling out potential induction of a malignant process. A similar enhancement of implant size was exhibited by pig embryonic pancreas precursor tissue. The growth of pancreatic tissues was determined by monitoring blood levels of pig insulin using a specific ELISA, previously shown to correlate with implant size. Likewise, the total volume of insulin positive cells was 0.68 ± 0.21mm3 in Factor VIII NOD-SCID versus 0.23 ± 0.18mm3 in non-hemophilic NOD-SCID recipients, respectively, suggesting an overall enhancement of implant size by a factor of three (p<0.05). A significant enhancement, by at least a factor of two of implant size, was also found upon implantation of embryonic pig liver in Factor VIII KO NOD-SCID mice, as evaluated by ELISA for pig albumin blood levels.In order to rule out potential artifacts due to genetic abnormalities in NOD-SCID FVIII KO mice, we attempted to introduce the Factor VIII KO mutation into a different SCID mouse, namely, RAG−/−. Thus, a new RAG−/− FVIII KO colony was established and similar results of oversized implants were achieved. In contrast to the pig implants, organ size was not enhanced upon implantation of mouse embryonic tissue into Factor VIII KO SCID mice. The lack of any effect on mouse embryonic transplants suggests that the role of Factor VIII is likely limited to a checkpoint of excessive growth (i.e. overgrowth checkpoint) which only operates upon implantation of tissue from a larger animal, such as the pig. To test the potential control by Factor VIII of oversized organs in a more physiological setting, we further tested its role in controlling G-CSF mediated splenomegaly. Thus, treatment with G-CSF of Factor VIII KO C57BL/6 mice was associated with significantly enhanced splenomegaly, compared to that found in the non-hemophilic counterparts, upon infusion of the same doses of G-CSF. Average spleen weight in the former group was 271.03 ± 154.86 mg, compared to 182.5 ± 58.43 mg in the latter group, p = 0.022). Furthermore, continuous administration by osmotic pumps of exogenous Factor VIII inhibited the enhanced splenomegaly in SCID FVIII KO mice under G-CSF stimulation (P<0.033)). Considering that Factor VIII KO could potentially affect the activity of other factors along the coagulation cascade, we further interrogated the role of other factors in the two models for organ size control. Thus, we found that enoxaparin, which inhibits the activity of both factor Xa and thrombin, markedly enhances G-CSF splenomegaly (p = 0.0017), as well as the size of pig embryonic liver implants in SCID mice with wild type Factor VIII (p<0.05). Likewise, a direct thrombin inhibitor (dabigatran) was found to significantly enhance G-CSF splenomegaly (p=0.0004), suggesting that it might be mediated by thrombin downstream of Factor VIII. Indeed, apart from its role in clot formation, thrombin has been shown to be capable of signaling through Protease-Activated Receptors (PARs), which are expressed throughout the body and known to be involved in vascular responses, embryonic development and malignancies. Collectively, our surprising set of observations suggests a yet unknown role in organ size control for factors of the coagulation cascade.

Chronic insulitis after partial islet damage by passive insulin antibody transfer.

The effect of passive insulin antibody transfer into recipient mice was reevaluated. Mice of inbred strains BALB/c, C57Bl/6 and DBA/2 received 10 injections of hyperimmune guinea pig insulin antiserum within 2 weeks. Ten days later the animals were sacrificed. It was found that up to 40% of animals had developed chronic lymphocytic infiltrations of pancreatic islets (insulitis) without showing diabetic symptoms. It is concluded that repeated damaging of islet tissue by passive insulin antibody transfer may result in chronic, possibly autoimmune insulitis. Our observation supports the concept that partial damage of pancreatic islets by environmental factors may trigger an organ-specific progressive islet cell destruction process mediated by immune cells.

Deletion of the Gene Encoding the Ubiquitously Expressed Glucose-6-phosphatase Catalytic Subunit-related Protein (UGRP)/Glucose-6-phosphatase Catalytic Subunit-β Results in Lowered Plasma Cholesterol and Elevated Glucagon

Deletion of the Gene Encoding the Ubiquitously Expressed Glucose-6-phosphatase Catalytic Subunit-related Protein (UGRP)/Glucose-6-phosphatase Catalytic Subunit-β Results in Lowered Plasma Cholesterol and Elevated Glucagon

Hyperthermic preconditioning protects pig islet grafts from early inflammation but enhances rejection in immunocompetent mice.

The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43 degrees C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1beta, IFN-gamma, or TNF-alpha was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.

Comparative study of biological activity of insulins of lower vertebrates in the novel adenylyl cyclase test-system

The biological activity of insulins of lower vertebrates (teleosts— Oncorhynchus gorbuscha, Scorpaena porcus, chondrosteans— Acipenser guldenstaedti and cyclostomates— Lamperta fluviatilis) was studied and compared with that of standard pig insulin. The determination of biological activity was made using the novel adenylyl cyclase (AC) test-system based on the adenylyl cyclase signaling mechanism (ACSM) of insulin action discovered earlier by the authors. The biological activity of insulins was estimated as EC 50, i.e. concentration leading to half-maximal activating effect of the hormone (10 −11–10 −7 M), in vitro, on adenylyl cyclase in two types of the target tissues: in membrane fractions of the muscles of rat and mollusc Anodonta cygnea. In rat, the efficiency of insulins was found to decrease in the following order: pig insulin>scorpaena insulin>gorbuscha insulin>sturgeon insulin>lamprey insulin. In the mollusc, the order was different: sturgeon insulin>scorpaena insulin>pig insulin>gorbuscha insulin. Lamprey insulin at the same concentrations did not apparently reach the maximal adenylyl cyclase activating effect. The suggestion was made that differences in the biological activity of insulins depend on the hormone structure and a number of indexes characteristic of the adenylyl cyclase test-system in the vertebrate and invertebrate tissues. The proposed adenylyl cyclase test-system is highly sensitive to insulin at physiological concentrations, has good reproduction and is easy to apply.

Three-beam interference is a sensitive measure of the efficacy of macromolecular refinement techniques.

Triplet phases recorded from insulin crystals were used to measure the improvement of phases during model refinement and to quantify the contribution made by each step in the refinement. Conventional amplitude data were recorded to 1.5 A resolution from rhombohedral pig insulin crystals using 1.54 A Cu Kalpha radiation. An initial atomic model and starting phases were obtained from a published structure and the atomic model was refined against the amplitude data using CNS. The refined phases were compared with 800 triplet phases that were measured from similar crystals using a three-beam interference technique and 1.1 A wavelength synchrotron radiation. The solvent region was improved further using a novel density-modification procedure. Calculated triplet phases were obtained from the model after each step in the refinement and were compared with the recorded triplet phases. The average difference between the recorded triplet phases and the calculated triplet phases was used as an unbiased measure of the correctness of the model at each stage in the refinement. The average individual phase error was estimated from discrepancies from triplet phases after each refinement step. Conventional atomic refinement of an approximate starting model reduced the average individual phase error from 21.6 to 14.7 degrees. Improvement of the solvent region, including the difference-map flattening procedure, reduced the individual phase error by a further 2.6 degrees. Modeling the discrete disorder of four amino acids accounted for an additional 0.5 degrees improvement and the final individual phase error was 11.6 degrees.

Recombinant protein drugs

What has been the real contribution of molecular biology, i.e. recombinant DNA technology to human therapeutics? Like Brazil it will always have a great future! This book of essays on the first generation of protein drugs made by recombinant protein synthesis is a fascinating melange of gossip, science, history and opinion, covering the 20 or so years since human insulin was first introduced. Since then, 1982, we have had haemophiliac globulins, growth hormone, erythropoietin, colony stimulating factors, interferon and clot busters, all natural proteins or ‘first generation’ recombinant products. We can see how the sector has matured now that generic producers are setting up and much work has taken place to develop ‘improved’ synthetic versions of natural products. Now we look to a second generation of artificial or hybrid proteins such as immunocytokines or engineered thrombolytics. The essays on these subjects are all by distinguished participants in the various discovery/exploitation processes. This is recent history, sometimes told starchily, sometimes well. History always makes for a good read especially for those keen on repeating it. As usual, the path to new therapies is not a simple one. It started with technical difficulties and suffered the usual unexpected failures and compensatory successes. Who would have predicted that human insulin would be less well tolerated than pig insulin? Who would have seen recombinant erythropoietin as a ‘drug of abuse’ in the Tour de France? On the postitive side, the introduction of colony stimulating factors has improved the practice of oncology, enabling neutropenia induced by cytotoxic agents to be treated, allowing more effective chemotherapy. Likewise tissue-type plasminogen activator has been a significant improvement over streptokinase in the therapy of myocardial infarction although, not surprisingly, it is just as difficult to use successfully in stroke as streptokinase. Plasminogen activators have now been tinkered with to improve their performance in the same way as classical medicinal products for treatments in the conventional pharmaceutical arena. The next step in recombinant protein therapeutics is to construct hybrid therapeutic functions. This concept has not been a great success in medicinal chemistry with α/β−adrenoceptor blockers but hope springs eternal and the chapter on immunocytokines makes an interesting read. There is also a chapter on improving protein pharmacokinetics, a universal problem. Finally to generation 3, gene therapy. Here the review by Friedman exhorts us that ‘definitive control of human disease is now in our grasp – loosely but without too much doubt.’ A ringing endorsement? So have the last 20 years seen recombinant technology revolutionize therapeutics? There have been big successes, both medical and financial. Standing back from the hype it seems, nonetheless, that a rather slight collection of therapies have, as yet, been brought forth. Certainly all the recombinant proteins combined would not equal the therapeutic worth of vaccines or antibiotics. The more major contribution of recombinant DNA technology to therapeutics must surely be its role in the discovery process for conventional chemical drugs. The chemical plant seems pretty likely to survive a long while yet.

Ethical challenges of xenotransplantation

The negative aspects of xenotransplantation are the high risk of organ rejection, which is greater when the genetic difference between animal and man is higher, as well as the transmission to the human being of zoonoses and retrovi- ruses that are potentially lethal, although they could be harmless in the animal donor. The risk of xenozoonoses to recipients and the general population is currently the main issue concerning the ethics of xenotransplantation. 2 Cur- rently, the transgenic pig is the most adequate species for organ donation and xenotransplantation. It breeds rapidly, its organs present approximately the same size and mor- phology as human organs, it is not endangered, it has been used for centuries in human alimentation, and pig heart valves and insulin have been used for several decades without problems. 2 Several studies are being conducted to assess the risk of transmission of infections from transgenic pigs to man. 3 The use of organs from nonhuman primates, although they pose a very low risk of hyperacute rejection (due to their phylogenetic closeness to humans), is re- stricted because of their slow breeding, the high costs involved in breeding them in a pathogen-free environment, the variability in the size of their organs, and the opposition of several animal rights activists like Peter Singer. 4 Further- more, the risk of transmission of microorganisms patho- genic to the human species is also higher and could lead to epidemics. 2 In our opinion, the clinical trials with porcine xenotrans- plants in human beings is ethically advisable despite their potential risks. The progress of medicine throughout the ages has always been associated with some degree of uncertainty and risk. As D. White says, if xenotransplan- tation can only be introduced into the clinic under condi- tions of zero tolerance of failure, then it is destined never to become a clinical reality. 3 That is in accordance with the conclusions of the serious report of the Nuffield Council on Bioethics and other institutions. 3,5 It is essential that the human recipients of xenotrans- plants give their informed consent to this new procedure, which excludes the involvement of those below the age of consent. They must be made aware of the risks involved, namely the possible transmission of infections, the potential benefits, and alternatives available. On the other hand, all recipients should be followed up closely. Furthermore, we propose that human clinical trials should start by gaining experience first, with the transplantation of organs such as the kidneys, before the use of vital organs such as the liver or heart. Therefore, we think that xenotransplantation, from an ethical point of view, represents a valid and defensible alternative to face the problem of the increasing shortage of organs available for transplantation. Meanwhile, we wait with interest for the results of the studies already in progress.