Amyloid formation in human islets is enhanced by heparin and inhibited by heparinase.

Islet transplantation is a promising therapy for patients with diabetes, but its long-term success is limited by many factors, including the formation of islet amyloid deposits. Heparin is employed in clinical islet transplantation to reduce clotting but also promotes fibrillization of amyloidogenic proteins. We hypothesized that heparin treatment of islets during pre-transplant culture may enhance amyloid formation leading to beta cell loss and graft dysfunction. Heparin promoted the fibrillization of human islet amyloid polypeptide (IAPP) and enhanced its toxicity to INS-1 beta cells. Heparin increased amyloid deposition in cultured human islets, but surprisingly decreased islet cell apoptosis. Treatment of human islets with heparin prior to transplantation increased the likelihood of graft failure. Removal of islet heparan sulfate glycosaminoglycans, which localize with islet amyloid deposits in type 2 diabetes, by heparinase treatment decreased amyloid deposition and protected against islet cell death. These findings raise the possibility that pretransplant treatment of human islets with heparin could potentiate IAPP aggregation and amyloid formation and may be detrimental to subsequent graft function.

The c.7409G>A (p.Cys2470Tyr) Variant of FBN1: Phenotypic Variability across Three Generations.

Marfan syndrome is an autosomal dominant connective tissue disorder caused by mutations in the fibrillin gene FBN1, which encodes an extracellular matrix glycoprotein. Major features of Marfan syndrome occur in the ocular, cardiovascular, and skeletal systems as well as in the dura mater. Approximately 60% of known disease-causing mutations are missense mutations of single amino acid residues. Effects on the cardiovascular system are classically associated with mutations in exons 24-32 of the 65 FBN1 exons and many, though not all, reports associate missense mutations in exons 59-65 with a mild cardiovascular phenotype. Here we present 5 related individuals among whom a c.7409G>A (p.Cys2470Tyr) missense variant in exon 59 of FBN1 is associated with significant cardiovascular features. The index case also had an apparently de novo 46,XX,del(5)(q33.1q33.3) deletion on chromosome 5. This family demonstrates skeletal, dermatological and neurological features consistent with Marfan syndrome but lacks significant ophthalmological findings to date. These findings suggest that FBN1 C-terminal missense mutations may not confer the ophthalmological features of Marfan syndrome, but they also confer a more significant risk for cardiovascular pathology than that suggested by previous studies. Furthermore, clinical data from this family supports the previously reported association of dural ectasia with C-terminal mutations.

Metabolic stress, IAPP and islet amyloid.

Amyloid forms within pancreatic islets in type 2 diabetes from aggregates of the ß-cell peptide islet amyloid polypeptide (IAPP). These aggregates are toxic to ß-cells, inducing ß-cell death and dysfunction, as well as inciting islet inflammation. The ß-cell is subject to a number of other stressors, including insulin resistance and hyperglycaemia, that may contribute to amyloid formation by increasing IAPP production by the ß-cell. ß-Cell dysfunction, evident as impaired glucose-stimulated insulin secretion and defective prohormone processing and exacerbated by metabolic stress, is also a likely prerequisite for islet amyloid formation to occur in type 2 diabetes. Islet transplants in patients with type 1 diabetes face similar stressors, and are subject to rapid amyloid formation and impaired proinsulin processing associated with progressive loss of ß-cell function and mass. Declining ß-cell mass is predicted to increase metabolic demand on remaining ß-cells, promoting a feed-forward cycle of ß-cell decline.

Reverse engineering an amyloid aggregation pathway with dimensional analysis and scaling.

Human islet amyloid polypeptide (hIAPP) is a cytotoxic protein that aggregates into oligomers and fibrils that kill pancreatic ß-cells. Here we analyze hIAPP aggregation in vitro, measured via thioflavin-T fluorescence. We use mass-action kinetics and scaling analysis to reconstruct the aggregation pathway, and find that the initiation step requires four hIAPP monomers. After this step, monomers join the nucleus in pairs, until the first stable nucleus (of size approximately 20 monomers) is formed. This nucleus then elongates by successive addition of single monomers. We find that the best-fit of our data is achieved when we include a secondary fibril-dependent nucleation pathway in the reaction scheme. We predict how interventions that change rates of fibril elongation or nucleation rates affect the accumulation of potentially cytotoxic oligomer species. Our results demonstrate the power of scaling analysis in reverse engineering biochemical aggregation pathways.

Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts.

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.

Toxic oligomers and islet beta cell death: guilty by association or convicted by circumstantial evidence?

Type 2 diabetes is a progressive disease characterised by islet amyloid deposits in the majority of patients. Amyloid formation is considered a significant factor in deterioration of islet function and reduction in beta cell mass, and involves aggregation of monomers of the normally soluble beta cell peptide, human islet amyloid polypeptide (hIAPP) into oligomers, fibrils and, ultimately, mature amyloid deposits. Despite extensive in vitro studies, the process of hIAPP aggregation in vivo is poorly understood, though it is widely reported to promote cytotoxicity. Recently, studies have suggested that only the early stages of fibril assembly, and in particular small hIAPP oligomers, are responsible for beta cell cytotoxicity. This challenges the prior concept that newly formed fibrils and/or mature fibrillar amyloid are cytotoxic. Herein, evidence both for and against the toxic hIAPP oligomer hypothesis is presented; from this, it is apparent that what exactly causes beta cell death when hIAPP aggregates remains debatable. Moreover, substantially more work with more specific reagents and techniques than are currently available will be required to identify conclusively the toxic species resulting from hIAPP aggregation. Keeping an open mind on the nature of the cytotoxic insult has implications for therapeutic developments and clinical care in type 2 diabetes.

Potential of selective insecticides for managing Uraba lugens (Lepidoptera: Nolidae) on Eucalypts.

The leaf skeletonizer Uraba lugens Walker (Lepidoptera: Nolidae), an Australian species, locally known as "gumleaf skeletonizer", is well established in New Zealand. This insect has the potential to become a serious pest of forestry and amenity eucalypts (Eucalyptus spp.) and is the focus of a long-term management program. The use of synthetic chemical or biological insecticides is one possible control method within an integrated control program. A series of dose-response trials were conducted using laboratory bioassays to test the efficacy of several insecticides against U. lugens: pyrethroids, spinosad, Bacillus thuringiensis kurstaki Berliner (Btk) and an insect growth regulator, Mimic. Pyrethroids and spinosad proved highly effective against U. lugens larvae, achieving 100% mortality after 3-6-d exposure. The performance of Btk was lower against gregarious skeletonizing larvae compared with solitary chewing larvae. When good coverage of the target foliage is achieved, >90% mortality is possible with Btk. Mimic performed poorly against U. lugens compared with other insecticides tested (<60% mortality). The Eucalyptus species on which larvae were feeding significantly altered insecticide efficacy. Treatments applied to Eucalyptus nitens (Deane & Maiden) Maiden had reduced efficacy compared with E. cinerea F. Muell. ex Benth. or E. fastigata Deane & Maiden. Cooler temperatures also reduced insecticide efficacy, presumably by decreasing movement and food consumption by U. lugens. Recommendations on spray applications to control U. lugens in New Zealand are given.

Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic yeast Pichia pastoris.

A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to achieve high cell density. Induction times were short to maximize rBoNTA(H(C)) production while minimizing proteolytic degradation. Concentration of rBoNTA(H(C)) in yeast cell lysates was generally 1-2% of the total protein based on ELISA analysis. The H(C) fragment was purified from cell lysates using a multistep ion-exchange (IEC) chromatographic process, including SP, Q, and HS resins. The zwitterionic detergent Chaps was included in the buffer system to combat possible interactions, such as protein-protein or protein-DNA interactions. Following IEC was a hydrophobic interaction chromatography (HIC) polishing step, using phenyl resin. The H(C) fragment was purified to >95% purity with yields up to 450 mg/kg cells based on ELISA and Bradford protein assay. The purified H(C) fragment of serotype A was stable, elicited an immune response in mice, and was protected upon challenge with native botulinum type A neurotoxin.

Production and purification of the heavy-chain fragment C of botulinum neurotoxin, serotype B, expressed in the methylotrophic yeast Pichia pastoris.

A recombinant Hc fragment of botulinum neurotoxin, serotype B (rBoNTB(Hc)), has been successfully expressed in a Mut+ strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. The fermentation process consisted of batch phase on glycerol, followed by glycerol and methanol fed-batch phases yielding a final cell mass of 60 g/L (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure Hc fragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified Hc fragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin.

Separation of estrogens and rodenticides using capillary electrophoresis with aqueous-methanolic buffers.

Capillary electrophoresis (CE) has proven to be an efficient method for the separation of various charged and neutral analytes. For analytes having limited solubility in water, the CE mode of separation has been micellar electrokinetic capillary chromatography (MECC). However, another approach is the direct addition of an organic solvent to a non-MECC CE separation system. Walbroehl and Jorgenson and also Balchunas and Sepaniak have reported on the use of organics in CE but the focus of their work was using MECC to separate small organic compounds. This work examines the use of aqueous-methanolic buffers in non-MECC CE separations of estrogens and rodenticides.