Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of ¹²⁵I-insulin and of insulin-related factor-1 (¹²⁵I-IGF-1) by plasma membranes of the rat liver and brain. The IC₅₀ value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (10⁴ ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC₅₀ value in the case of ¹²⁵I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 10⁴ ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.     

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.     

The mechanisms of fatty-acid inhibition of electron transport in chloroplasts

Unsaturated fatty acids at concentrations of 1–2 μmol mg^-1 chlorophyll decrease the intensity of long-lived delayed fluorescence and inhibit the Hill reaction in Pisum sativum L. chloroplasts in a pH-dependent and reversible manner. A charged form of the fatty acids is two times more effective than an undissociated form. Fatty acids, anionic and cationic detergents and urea inhibit activity and decrease the temperature of heat inactivation of the water-spilitting system. Sucrose at a concentration of 2.5 M protects chloroplasts against the effects of these compounds. It is concluded that their action can be explained by the denaturation of the water-splitting protein.     

Cold acclimation and photoinhibition of photosynthesis in Scots pine

Cold acclimation of Scots pine did not affect the susceptibility of photosynthesis to photoinhibition. Cold acclimation did however cause a suppression of the rate of CO₂ uptake, and at given light and temperature conditions a larger fraction of the photosystem II reaction centres were closed in cold-acclimated than in nonacclimated pine. Therefore, when assayed at the level of photosystem II reaction centres, i.e. in relation to the degree of photosystem closure, cold acclimation caused a significant increase in resistance to photoinhibition; at given levels of photosystem II closure the resistance to photoinhibition was higher after cold acclimation. This was particularly evident in measurements at 20° C. The amounts and activities of the majority of analyzed active oxygen scavengers were higher after cold acclimation. We suggest that this increase in protective enzymes and compounds, particularly Superoxide dismutase, ascorbate peroxidase, glutathione reductase and ascorbate of the chloroplasts, enables Scots pine to avoid excessive photoinhibition of photosynthesis despite partial suppression of photosynthesis upon cold acclimation. An increased capacity for light-induced de-epoxidation of violaxanthin to zeaxanthin upon cold acclimation may also be of significance.Abbreviations APX ascorbate peroxidase - DHA dehydroascorbate - DHAR dehydroascorbate reductase - Fm maximal fluorescence when all reaction centres are closed - Fv/Fm maximum photochemical yield of PSII - GR glutathione reductase - GSH reduced glutathione - Je rate of photosynthetic electron transport - MDAR monodehydroascorbate reductase - q_N nonphotochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - SOD superoxide dismutase This work was supported by the Swedish Natural Science Research Council and the National Natural Science Foundation of China.     

Regular proteinaceous layers ofThermococcus stetteri cell envelope

Proteinaceous layers of theThermococcus stetteri cell envelope were investigated and found to consist of regularly arrayed subunits 18 nm in diameter. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two major proteins were present. They were glycosylated and had molecular weights of 80,000 and 210,000. In addition to two external regular proteinaceous layers, cells ofT. stetteri were found to have internal regular layers tightly attached to the cytoplasmic membrane. In the region of flagella attachment to the cell, polar membrane-like structures were found in the cytoplasm.     

Changes in antioxidants and kinetics of glutathione-S-transferase of maize in response to isoproturon treatment

Abstract

Isoproturon at the recommended field dose (RFD) significantly reduced fresh and dry weights of shoots and roots as well as chlorophyll and carotenoid contents of 10-day-old maize seedlings during the following 20 days. The higher the herbicide dose, the greater the reduction. Meanwhile, ascorbate (AsA) and reduced glutathione (GSH) increased in leaves for only the first few days. Similar increases in activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) were detected. Low doses caused general increases while high doses induced diminutions; however, CAT and APX activities were inhibited by all doses. Nevertheless, H₂O₂ was significantly accumulated throughout the experiment; the magnitude of accumulation increased with time and herbicide dose. On the contrary, there were significant inhibitions in activities of the glutathione S-transferase (GST) isoforms (GST_(CDNB), GST_(ALA), or GST_(MET)) with no variation in GST_(ATR); the inhibition was greater with increasing isoproturon doses. These findings suggest the occurrence of an oxidative stress induced by isoproturon, a state that prolonged with increasing herbicide dose and/or treatment time. Moreover, V _max of GST was lowered by isoproturon, whereas K _m was unchanged, indicating that the herbicide is a competitive inhibitor of GST.     

The FTO A/T Polymorphism and Elite Athletic Performance: A Study Involving Three Groups of European Athletes

Objective

The FTO A/T polymorphism (rs9939609) is a strong candidate to influence obesity-related traits. Elite athletes from many different sporting disciplines are characterized by low body fat. Therefore, the aim of this study was to assess whether athletic status is associated with the FTO A/T polymorphism.

Subjects and Methods

A large cohort of European Caucasians from Poland, Russia and Spain were tested to examine the association between FTO A/T polymorphism (rs9939609) and athletic status. A total of 551 athletes were divided by type of sport (endurance athletes, n = 266 vs. sprint/power athletes, n = 285) as well as by level of competition (elite-level vs. national-level). The control group consisted of 1,416 ethnically-matched, non-athletic participants, all Europeans. Multinomial logistic regression analyses were conducted to assess the association between FTO A/T genotypes and athletic status/competition level.

Results

There were no significantly greater/lesser odds of harbouring any type of genotype when comparing across athletic status (endurance athletes, sprint/power athletes or control participants). These effects were observed after controlling for sex and nationality. Furthermore, no significantly greater/lesser odds ratios were observed for any of the genotypes in respect to the level of competition (elite-level vs. national-level).

Conclusion

The FTO A/T polymorphism is not associated with elite athletic status in the largest group of elite athletes studied to date. Large collaborations and data sharing between researchers, as presented here, are strongly recommended to enhance the research in the field of exercise genomics.     

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)₃ or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.