Molecular Basis of Pathogenesis of Diabetes Mellitus Type 2- a New Perspective

Decreased insulin secretion due to beta cell dysfunction of the pancreas and defective utilization of insulin due to insulin resistance / Hyperinsulinemia are two important issues in the pathogenesis of DM2. There are many explanations in the literature to account for these two observed phenomena and their interrelationship. DM2 is believed to occur due to a complex interplay of environmental andBehavioural factors in genetically predisposed persons. Among the prominent theories explaining the pathogenesis of DM2, the viscera- Portal hypothesis, the Ectopic fat hypothesis and the adipose tissue as an endocrinal gland are prominent. Besides, the role played by oxidative stress, metabolic stress, mitochondrial dysfunction, endoplasmic reticulum stress, etc. are also advanced. It is felt that basic to and at the core of all the observed facts, is the shift of energy metabolism from normal glycolysis to B- oxidation of fats. Hence, how B - oxidation prevails over glycolysis is the fundamental issue to be addressed together with its interrelationships with insulin resistance, as to which is the cause and which is the effect. At the molecular level, an attempt to find answers to the above questions is made in this paper.To this extent, the Randle fatty acid cycle (Substrate competition theory of Randle) is suitably modified and applied to explain the switch of Energy metabolisms in DM2 .Defective disulfide bond formation of the insulin receptor which makes it physiologically ineffective, is suggested as the cause of the insulin resistance where as the prevailing molecular mechanisms stress on post-receptor signaling defect. The cause and effect of both are discussed. This line is considered to be a departure from traditional approaches broached above and briefly outlined in this article.

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A-C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A-C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Role of the Trp-disulfide Triads in the UV Light Induced Degradation of a Monoclonal Antibody scFv.

Proteins are targets for photodegradation due to absorption of incident light by endogenous chromophores, e.g aromatic side chains. In this work we study the role of Trp-disulfide triads in the light induced loss of immunoglobulin activity. Study Design: We investigated a single chain variable fragment (scFv) of the Trp-disulfide triad containing monoclonal antibody 82D6A3. The scFv binds to von Willebrand factor (VWF) and upon illumination with near UV-B-light the scFv partially loses its binding capacity to VWF. In order to relate this observed degeneration to the specific Trp-disulfide triads, we mutated W35(VL) and W36(VH) which are in direct contact with the disulfide

Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.(AU)

Isolation,Purification and Identification of Recombinant Human Hepcidin / 微生物学通报

Method of isolation and purification of recombinant hepcidin was described, and the bioactivity of the protein was assayed in this paper.The oxidation of his-hepcidin was carried out in the cysteine-cystine system, and the multimers were removed through gel filtration under denaturation condition.Then the protein was refolded by continuous dilution and digested by enterokinase.The total yield of his-hepcidin before enterokinase cleavage is 50%, and the purity is above 95%.Through agar diffusion assay, the recombinant hepcidin displayed obvious antibacterial activity against B.subtilis.The LC-ESI-MS analysis of recombinant hepcidin showed that the measured molecular weight accorded with the calculated molecular weight, and the CD spectrum indicated that the secondary structure of recombinant hepcidin is similar with native hepcidin.