Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis.     

Studies on the biology of Argas (A.) reflexus (Fabricius, 1794) (Acari: Ixodida: Argasidae). 2. Effect of alternating temperatures on embryonic development and egg hatch.

The paper presents the results of observations on the effect of temperature alterations between 9 degrees C and 30 degrees C every 6 and 12 hours, respectively, on the embryonic development and egg hatch of Argas (A.) reflexus. No effect of the frequency of temperature changes on the percentage of egg mortality, embryo mortality, abnormal egg hatching, or egg hatching into morphologically normal larvae was observed. The experiments showed that in changes temperature have a particularly detrimental effect on the eggs prior to blastulation.     

Albumin is a redox-active crowding agent that promotes oxidative folding of cysteine-rich peptides

Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin, and polysaccharides, influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13 residues (1.4 kDa) to 58-residues (6.5 kDa): conotoxins: GI, PVIIA, r11a, and bovine pancreatic trypsin inhibitor. Our results indicate that the excluded volume effect does not significantly alter the folding rates nor equilibria for these peptides. In stark contrast, folding reactions were dramatically accelerated, when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone was as effective as glutathione in promoting the oxidative folding of GI conotoxin at concentrations typically found in the ER. To the best of our knowledge, this is the first report and quantitative characterization of oxidative folding of peptides mediated by other than thioredoxin-based protein disulfide bonds. Our work raises a possibility that concurrent secretory and ER-resident proteins may influence the oxidative folding of small, cysteine-rich peptides not as crowding agents, but as redox-active factors.     

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Mental patients in prisons

Mental conditions usually affect cognitive, emotional and volitional aspects and functions of the personality, which are also functions of interest in law, as they are essential at the time of adjudicating guilt, labeling the accused a criminal, and proffering a sentence. A relationship between mental illness and criminality has, thus, been described and given as one of the reasons for the large number of mental patients in prisons. Whether this relationship is one of causality or one that flows through many other variables is a matter of debate, but there is no debating that prisons have become a de facto part, and an important one, of mental health systems in many countries. This paper deals with the issue of the relationship and provides estimates of prevalence of mental patients in prisons culled from many studies in different countries. It also provides some direction for the management of mental patients as they crowd correctional systems.     

Estimation of formamide harmonic and anharmonic modes in the Kohn-Sham limit using the polarization consistent basis sets

Formamide harmonic and anharmonic frequencies of fundamental vibrations in the gas phase and in several solvents were successfully estimated in the B3LYP Kohn-Sham complete basis set limit (KS CBS). CBS results were obtained by extrapolating a power function (two-parameter formula) to the results calculated with polarization-consistent basis sets. Anharmonic corrections using the second order perturbation treatment (PT2) and hybrid B3LYP functional combined with polarization consistent pc-n (n = 0, 1, 2, 3, 4) and several Pople’s basis sets were analyzed for all fundamental formamide vibrational modes in the gas phase and solution. Solvent effects were modeled within a PCM method. The anharmonic frequency of diagnostic amide vibration C = O in the gas phase and the CCl₄ solution calculated with the VPT2 method was significantly closer to experimental data than the corresponding harmonic frequency. Both harmonic and anharmonic frequencies of C = O stretching mode decreased linearly with solvent polarity, expressed by relative environment permittivity (ε) ratio (ε − 1)/(2ε + 1). However, an unphysical behavior of solvent dependence of some low frequency anharmonic amide modes of formamide (e.g., CN stretch, NH₂ scissoring, and NH₂ in plane bend) was observed, probably due to the presence of severe anharmonicity and Fermi resonance.     

MHC influences infection with parasites and winter survival in the root vole Microtus oeconomus

Selective pressure from parasites is thought to maintain the polymorphism of major histocompatibility complex (MHC) genes. Although a number of studies have shown a relationship between the MHC and parasitic infections, the fitness consequences of such associations are less well documented. In the present paper, we characterised the variation in exon 2 of MHC class II DRB gene in the root vole and examined the effects of that gene on parasite prevalence and winter survival. We identified 18 unique exon 2 sequences, which translated into 10 unique amino acid sequences. Phylogenetic analysis revealed the presence of three distinct clusters, and allele distributions among these individuals suggested that the clusters correspond to three different loci. Although the rate of synonymous substitutions (d_S) exceeded the rate of nonsynonymous substitutions (d_N) across sequences, implying purifying selection, d_N was significantly elevated at antigen-binding sites, suggesting that these sites could be under positive selection. Screening for parasites revealed a moderate prevalence of infection with gastrointestinal parasites (24 % infected), but a high infection rate for blood parasites (56 % infected). Infection with the blood parasite Babesia ssp. decreased survival almost twofold (25.7 vs. 13.9 %). Animals possessing the amino acid sequence AA*08 survived better than others (44.9 vs. 22 %), and they were infected with Babesia ssp. less often (13.9 vs 25.7 %). In contrast, individuals carrying allele AA*05 were infected more often (31.7 vs. 15.3 %). Heterozygosity at one of the putative loci was associated with a lower probability of infection with Babesia ssp., but at the other locus, the association was reversed. The unexpected latter result could be at least partly explained by the increased frequency of the susceptible allele AA*05 among heterozygotes. Overall, we demonstrate that infection with Babesia ssp. is a strong predictor of winter survival and that MHC genes are important predictors of infection status as well as survival in the root vole.     

A possible Fourier transform infrared‐based plasma fingerprint of angiotensin‐converting enzyme inhibitor‐induced reversal of endothelial dysfunction in diabetic mice

Angiotensin‐converting enzyme inhibitors (ACE‐I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)‐based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased β‐sheet formation. Spectral markers at 3010, 1520/1238 cm^?1, representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE‐I.