Effects of pyridoxine supplementation on blood glucocorticoids level, aspartate aminotransferase activity and glucose tolerance pattern under acute hypoxic stress

Studies were conducted on male adult rabbits to find out the changes in blood glucocorticoid levels along with the changes in aspartate aminotransferase activity in blood and the role of pyridoxine on the glucose tolerance pattern under hypoxic stress. Hypoxic stress was produced by exposing the animals to a simulated altitude of 7,000 m for 6 h. In the first set of experiments 10 rabbits were used. Blood haemoglobin level, plasma and erythrocyte glucocorticoid levels and erythrocyte GOT activity were measured just before and after the exposure to hypoxia. Erythrocyte GOT activity was measured both without and with 50 mg of pyridoxal phosphate addition to the incubation mixture. Glucocorticoid levels in plasma increased by 11% whereas in erythrocytes the increase was 55% after hypoxia. Percent stimulation of erythrocyte GOT activity with pyridoxal phosphate before exposure to hypoxia was 180% but increased to 321% after exposure. In the second set of experiments another 10 rabbits were used. First they were exposed to hypoxia without pyridoxine hydrochloride feeding and then after 7 days with 3 mg of pyridoxine hydrochloride feeding. For glucose tolerance tests the animals were fed with 1 g of glucose immediately after the hypoxic exposures. Plasma reduced glutathione (GSH), LDH and ICDH activities increased and GOT activity was depressed after hypoxic stress, but when the animals were fed pyridoxine hydrochloride prior to the exposure the enzyme activities remained unaltered after hypoxic stress. Pyridoxine hydrochloride did not alter the pattern of glucose tolerance after hypoxic stress.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

The HPr proteins from the thermophile Bacillus stearothermophilus can form domain-swapped dimers

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B.subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.     

Cadmium effects on sperm morphology and semenogelin with relates to increased ROS in infertile smokers: An in vitro and in silico approach

Smoking releases cadmium (Cd), the metal toxicant which causes an imbalance in reactive oxygen species level in seminal plasma. This imbalance is envisaged to impair the sperm DNA morphology and thereby result in male infertility. In order to correlate this association, we performed in vitro and in silico studies and evaluated the influence of reactive oxygen species imbalance on sperm morphology impairments due to smoking. The study included 76 infertile smokers, 72 infertile non-smokers, 68 fertile smokers and 74 fertile non-smokers (control). Semen samples were collected at regular intervals from all the subjects. Semen parameters were examined by computer assisted semen analysis, quantification of metal toxicant by atomic absorption spectrophotometer, assessment of antioxidants through enzymatic and non-enzymatic methods, diagnosis of reactive oxygen species by nitro blue tetrazolium method and Cd influence on sperm protein by in vitro and in silico methods. Our analysis revealed that the levels of cigarette toxicants in semen were high, accompanied by low levels of antioxidants in seminal plasma of infertile smoker subjects. In addition the investigation of Cd treated sperm cells through scanning electronic microscope showed the mid piece damage of spermatozoa. The dispersive X-ray analysis to identify the elemental composition further confirmed the presence of Cd. Finally, the in-silico analysis on semenogelin sequences revealed the D-H-D motif which represents a favourable binding site for Cd coordination. Our findings clearly indicated the influence of Cd on reactive oxygen species leading to impaired sperm morphology leading to male infertility.     

Structure and characterization of a high affinity C5a monoclonal antibody that blocks binding to C5aR1 and C5aR2 receptors

C5a is a potent anaphylatoxin that modulates inflammation through the C5aR1 and C5aR2 receptors. The molecular interactions between C5a–C5aR1 receptor are well defined, whereas C5a–C5aR2 receptor interactions are poorly understood. Here, we describe the generation of a human antibody, MEDI7814, that neutralizes C5a and C5adesArg binding to the C5aR1 and C5aR2 receptors, without affecting complement–mediated bacterial cell killing. Unlike other anti–C5a mAbs described, this antibody has been shown to inhibit the effects of C5a by blocking C5a binding to both C5aR1 and C5aR2 receptors. The crystal structure of the antibody in complex with human C5a reveals a discontinuous epitope of 22 amino acids. This is the first time the epitope for an antibody that blocks C5aR1 and C5aR2 receptors has been described, and this work provides a basis for molecular studies aimed at further understanding the C5a–C5aR2 receptor interaction. MEDI7814 has therapeutic potential for the treatment of acute inflammatory conditions in which both C5a receptors may mediate inflammation, such as sepsis or renal ischemia–reperfusion injury.     

Conformational analysis of thymidylate synthase from amino acid sequence and circular dichroism

Circular dichroism studies were carried out in the vacuum ultraviolet region for thymidylate synthase from Lactobacillus casei and its ligand complexes. The CD spectrum was analyzed for secondary structure by our method and the variable selection method, and both gave similar results. Our method predicts 33% alpha-helix, 25% (23% antiparallel and 2% parallel) beta-sheet, 20% turns, and 16% other structure. The secondary structure of this protein was also predicted from the amino acid sequence by four different methods. Though there is a variation in the prediction among these methods, the prediction of 32% alpha-helix and 23% beta-sheet by combining the four methods is in excellent agreement with our CD results. Further, the location of the predicted regions of alpha-helices and beta-strands along the sequence and the CD characteristics strongly suggest that this protein belongs to an alpha + beta structural class. Binding of the inhibitor FdUMP or the cofactor 5,10-methylenetetrahydrofolate did not change the CD spectrum. However, when both ligands were present, there was a significant change in the CD spectrum and the maximum changes occurred when the concentration of FdUMP was 1 mol/mol of enzyme. The addition of FdUMP and cofactor causes, respectively, a 5% and 6% decrease in beta-sheet and beta-turns and about an 8% increase in "other" structure.     

Thyroid gland function during cross adaptation to heat and cold in man

Plasma thyroxine (T₄), triiodothyronine (T₃) and thyrotropin (TSH) levels were monitored in 10 healthy euthyroid male subjects of the age group 20 to 30 years before and during heat and cold acclimatisation schedule in a sequential manner. The subjects were exposed to 45C DB and 30% relative humidity in a hot chamber for 2 hours daily for 8 consecutive days. Subsequently they were exposed to cold for 4 hours daily at 10C for 21 days. The mean plasma T₄ and T₃ concentration before exposure to heat were 7.87±0.82 ug/dl and 159.8±9.1 ng/dl respectively. A significant decrease in both T₄ (p<0.05) and T₃ (p<0.01) levels to mean values of 6.4±0.76g/dl and 129±7.9 ng/dl was recorded on day 4 of exposure to heat. Further significant decrease (p<0.05) over the preceding T₃ levels was observed on day 8 of heat exposure. Plasma T₄ and T₃ on day 21 of cold exposure was not significantly different from the levels reckoned after last day of heat exposure but was significantly lower than the pre-exposure values. Throughout the thermal stress schedule there was no change in the TSH levels. These observations suggest that a decrease in thyroid hormone levels during exposure to heat might be an adaptive process which continues even during cold acclimatisation.     

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.