In Type 1 Diabetes a Subset of Anti-Coxsackievirus B4 Antibodies Recognize Autoantigens and Induce Apoptosis of Pancreatic Beta Cells

Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients’sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients’ sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

The status and conservation of the Cape Gannet Morus capensis

The Cape Gannet Morus capensis is one of several seabird species endemic to the Benguela upwelling ecosystem (BUS) but whose population has recently decreased, leading to an unfavourable IUCN Red List assessment. Application of ‘JARA’ (‘Just Another Red-List Assessment,’ a Bayesian state-space tool used for IUCN Red List assessments) to updated information on the areas occupied by Cape Gannets and the nest densities of breeding birds at their six colonies, suggested that the species should be classified as Vulnerable. However, the rate of decrease of Cape Gannets in their most-recent generation exceeded that of the previous generation, primarily as a result of large decreases at Bird Island, Lambert’s Bay, and Malgas Island, off South Africa’s west coast (the western part of their range). Since the 1960s, there has been an ongoing redistribution of the species from northwest to southeast around southern Africa, and ～70% of the population now occurs on the south coast of South Africa, at Bird Island in Algoa Bay, on the eastern border of the BUS. Recruitment rather than adult survival may be limiting the present population; however, information on the seabird’s demographic parameters and mortality in fisheries is lacking for colonies in the northern part of the BUS. Presently, major threats to Cape Gannet include: substantially decreased availability of their preferred prey in the west; heavy mortalities of eggs, chicks and fledglings at and around colonies, inflicted by Cape Fur Seals Arctocephalus pusillus and other seabirds; substantial disturbance at colonies caused by Cape Fur Seals attacking adult gannets ashore; oiling; and disease.     

Purification and characterization of amine oxidase from pea seedlings

A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.     

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.     

Proliferative generation of mammalian auditory hair cells in culture

Hair cell (HC) and supporting cell (SC) productions are completed during early embryonic development of the mammalian cochlea. This study shows that acutely dissociated cells from the newborn rat organ of Corti, developed into so-called otospheres consisting of 98% nestin (+) cells when plated on a non-adherent substratum in the presence of either epidermal growth factor (EGF) or fibroblast growth factor (FGF2). Within cultured otospheres, nestin (+) cells were shown to express EGF receptor (EGFR) and FGFR2 and rapidly give rise to newly formed myosin VIIA (+) HCs and p27(KIP1) (+) SCs. Myosin VIIA (+) HCs had incorporated bromodeoxyuridine (BrdU) demonstrating that they were generated by a mitotic process. Ultrastructural studies confirmed that HCs had differentiated within the otosphere, as defined by the presence of both cuticular plates and stereocilia. This work raises the hypothesis that nestin (+) cells might be a source of newly generated HCs and SCs in the injured postnatal organ of Corti.     

Nitrogenase activity of Herbaspirillum seropedicae grown under low iron levels requires the products of nifXorf1 genes

Herbaspirillum seropedicae strains mutated in the nifX or orf1 genes showed 90% or 50% reduction in nitrogenase activity under low levels of iron or molybdenum respectively. Mutations in nifX or orf1 genes did not affect nif gene expression since a nifH::lacZ fusion was fully active in both mutants. nifX and the contiguous gene orf1 are essential for maximum nitrogen fixation under iron limitation and are probably involved in synthesis of nitrogenase iron or iron-molybdenum clusters.