Dexamethasone-mediated regulation of death and differentiation of muscle cells. Is hydrogen peroxide involved in the process?

The hypothetical involvement of H2O2 in dexamethasone-mediated regulation of muscle cell differentiation and elimination was studied. Rat L6 myoblasts and mouse C2C12 satellite cells were chosen for acute (24 h) and chronic (5 or 10 day) experiments. Mitogenicity and anabolism were both affected by H2O2. Micromolar concentrations of H2O2 inhibited DNA while stimulating protein synthesis. At the millimolar level, H2O2 led to cell death by apoptosis.Synthetic glucocorticoi - dexamethasone (Dex) was shown to effect muscle cell fate similarly to H2O2. Chronic treatment with H2O2 or Dex dose-dependently accelerated either the formation of myotubes or cell elimination. Dex-induced cell death slightly differed from classical apoptosis and was featured by the symptoms of cell senescence such as extensive cytoplasm vacuolisation, accumulation of inclusion-bodies and lack of low molecular weight oligonucleosomal DNA fragmentation but chromatin condensation. Antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase) abrogated Dex-dependent cell death. We conclude that H2O2 directly influences myogenesis and muscle cell elimination. Moreover, H2O2 can be considered as the potent mediator of glucocorticoid-dependent effects on muscle cells.     

Analysis of serine proteinase-inhibitor interaction by alanine shaving

We analyzed the energetic importance of residues surrounding the hot spot (the P(1) position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P(1) position to methyl groups caused a decrease in Delta G(den) values up to 6.4 kcal mole(-1), it did not influence the overall conformation of the inhibitor. We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability. To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis. Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole(-1)) than chymotrypsin (decrease up to 3.5 kcal mole(-1)). In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects. However, when P(1)-neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain-main chain intermolecular hydrogen bonds. Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P(1)-S(1) electrostatic interaction.     

Conformational subspace in simulation of early-stage protein folding

A probability calculus was used to simulate the early stages of protein folding in ab initio structure prediction. The probabilities of particular phi and psi angles for each of 20 amino acids as they occur in crystal forms of proteins were used to calculate the amount of information necessary for the occurrence of given phi and psi angles to be predicted. It was found that the amount of information needed to predict phi and psi angles with 5 degrees precision is much higher than the amount of information actually carried by individual amino acids in the polypeptide chain. To handle this problem, a limited conformational space for the preliminary search for optimal polypeptide structure is proposed based on a simplified geometrical model of the polypeptide chain and on the probability calculus. These two models, geometric and probabilistic, based on different sources, yield a common conclusion concerning how a limited conformational space can represent an early stage of polypeptide chain-folding simulation. The ribonuclease molecule was used to test the limited conformational space as a tool for modeling early-stage folding.     

Seasonal changes in the thermoregulation of laboratory golden hamsters during acclimation to seminatural outdoor conditions

Proper adjustments of the thermoregulatory mechanisms ensure survival in the natural environment. In the present study, we tested the hypothesis that laboratory golden hamsters (Mesocricetus auratus) housed under seminatural outdoor conditions are able to acclimatize to daily and seasonal changes in the environment despite their long history of breeding in captivity. The animals experienced natural changes in the photoperiod and ambient temperature characteristic for central Poland. During experiments in the thermal gradient system, the daily rhythms of body temperature (measured as the temperature of brown adipose tissue, TBAT), preferred ambient temperature (PTa) and activity were measured in summer, autumn and spring. We found that mean TBAT was highest in autumn and least in summer, reflecting seasonal changes in the capacity for nonshivering thermogenesis (NST). In summer, TBAT followed the robust daily rhythm with the amplitude of 1.1+/-0.1 degrees C. This amplitude was depressed in autumn (0.2+/-0.1 degrees C) and partially restored in spring (0.4+/-0.1 degrees C). Seasonal changes in the daily amplitude of TBAT recorded during both transitional periods, i.e., in autumn and spring, seem to be associated with hamsters' hibernation. In autumn, mean daily PTa was lower than in summer and spring, indicating the lowering of a set point for core body temperature (Tb) regulation. Locomotor activity was much higher in spring than in summer and autumn, and it always predominated at night. We conclude that laboratory golden hamsters housed under seminatural conditions express daily and seasonal changes in the thermoregulatory mechanisms that, despite long history of breeding in captivity, enable proper acclimatization to seasonally changing environment and ensure successful hibernation and winter survival.     

rep-PCR fingerptinting as a tool for the analysis of genomic diversity in Escherichia coli strains isolated from an aqueous/freshwater environment

The rep-PCR fingerprinting method, with the support of ERIC and REP primers, was used to analyse the genomic diversity of 93 E. coli strains isolated from lake water samples drawn at two different depths. The applied UPGMA for DNA analysis did not reveale any genomic similarities between the 48 E. coli strains derived from the subsurface-zone water and the 43 of the bottom-zone water. The considerable genomic diversity of the E. coli of the surface zone was expressed as a dendrogram in the form of 8 similarity groups comprising strains isolated from samples drawn over one month. The bottom-zone strains, which display a lesser degree of genomic diversity (5 similarity groups), showed distinct common features in their DNA fingerprints. In the similarity dendrogram for the bottom-zone, strains derived in different months of sampling were segregated into the same similarity groups. Applying REP primers in rep-PCR generates more complex fingerprints increasing the discriminatory power of the analysis, whereas the ERIC primer generates less complex fingerprint patterns, and is thus clearer to interpret.     

The Comparison of β-Thymosin Homologues Among Metazoa Supports an Arthropod-Nematode Clade

The definition of an Ecdysozoa clade among the protostomians, including all phyla with a regularly molted α-chitin-rich cuticle, has been one of the most provocative hypotheses to arise from recent investigations on animal phylogeny. Here we present evidence in favor of an arthropod-nematode clade, from the comparison of β-thymosin homologues among the Metazoa. Arthropods and nematodes share the absence of the highly conserved β-thymosin form found in all other documented bilaterian phyla as well as sponges, and the possession of a very unusual, internally triplicated homologue of the β-thymosin protein, unknown in other phyla. We argue that such discrete molecular character is phylogenetically very powerful and provides strong evidence for the monophyly of an arthropod-nematode clade. Received: 17 December 1999 / Accepted: 7 July 2000     

On estimating the probability of aperiodic outbursts of microbial populations from their fluctuating counts

The irregular sequence of counts of a microbial population, in the absence of observable corresponding environmental changes (e.g., temperature), can be regarded as reflecting the interplay of several unknown or random factors that favor or inhibit growth. Since these factors tend to balance one another, the fluctuations usually remain within bounds, and only by a coincidence—when all or most act in unison—does an ‘outburst’ occur. This situation can be represented mathematically as a sequence of independent random variables governed by a probability distribution. The concept was applied to reported microbial counts of ground meat and wastewater. It is found that the lognormal distribution could serve as a model, and that simulations from this model are indistinguishable from actual records. The parameters of the lognormal (or other) distribution can then be used to estimate the probability of a population outburst, i.e., an increase above a given threshold. Direct estimation of the outburst probability based on frequency of occurrence is also possible, but in some situations requires an impractically large number of observations. We compare the efficiency of these two methods of estimation. Such methods enable translation of irregular records of microbial counts into actual probabilities of an outburst of a given magnitude. Thus, if the environment remains ’stable’ or in dynamic equilibrium, the fluctuations should not be regarded merely as noise, but as a source of information and an indicator of potential population outbursts even where obvious signs do not exist.     

CD8+ T cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections

A protective role for CD8+ T cells during viral infections is generally accepted, but little is known about how CD8+ T cell responses develop during primary infections in infants, their efficacy, and how memory is established after viral clearance. We studied CD8+ T cell responses in bronchoalveolar lavage (BAL) samples and blood of infants with a severe primary respiratory syncytial virus (RSV) infection. RSV-specific CD8+ T cells with an activated effector cell phenotype: CD27+CD28+CD45RO+CCR7-CD38+HLA-DR+Granzyme B+CD127- could be identified in BAL and blood. A high proportion of these CD8+ T cells proliferated and functionally responded upon in vitro stimulation with RSV Ag. Thus, despite the very young age of the patients, a robust systemic virus-specific CD8+ T cell response was elicited against a localized respiratory infection. RSV-specific T cell numbers as well as the total number of activated effector type CD8+ T cells peaked in blood around day 9-12 after the onset of primary symptoms, i.e., at the time of recovery. The lack of a correlation between RSV-specific T cell numbers and parameters of disease severity make a prominent role in immune pathology unlikely, in contrast the T cells might be involved in the recovery process.     

Stabilizing effect of Zn2+ in native bovine rhodopsin

Single-molecule force spectroscopy (SMFS) is a powerful tool to dissect molecular interactions that govern the stability and function of proteins. We applied SMFS to understand the effect of Zn2+ on the molecular interactions underlying the structure of rhodopsin. Force-distance curves obtained from SMFS assays revealed the strength and location of molecular interactions that stabilize structural segments within this receptor. The inclusion of ZnCl2 in SMFS assay buffer increased the stability of most structural segments. This effect was not mimicked by CaCl2, CdCl2, or CoCl2. Thus, Zn2+ stabilizes the structure of rhodopsin in a specific manner.     

Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy

Transformation of a transected axonal tip into a growth cone (GC) is a critical step in the cascade leading to neuronal regeneration. Critical to the regrowth is the supply and concentration of vesicles at restricted sites along the cut axon. The mechanisms underlying these processes are largely unknown. Using online confocal imaging of transected, cultured Aplysia californica neurons, we report that axotomy leads to reorientation of the microtubule (MT) polarities and formation of two distinct MT-based vesicle traps at the cut axonal end. Approximately 100 microm proximal to the cut end, a selective trap for anterogradely transported vesicles is formed, which is the plus end trap. Distally, a minus end trap is formed that exclusively captures retrogradely transported vesicles. The concentration of anterogradely transported vesicles in the former trap optimizes the formation of a GC after axotomy.