A prevalent POLG CAG microsatellite length allele in humans and African great apes

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase (POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)₁₀ allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee (Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.     

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

Effects of peptide molecular mass and PEG chain length on the vasoreactivity of VIP and PACAP(1-38) in pegylated phospholipid micelles

Bioactive properties of certain amphipathic peptides are amplified when self-associated with sterically stabilized micelles (SSM) composed of polyethylene glycol (PEG)-conjugated phospholipids. The purpose of this study was to determine the effects of amphipathic peptide molecular mass and PEG chain length on vasoreactivity evoked by vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, and pituitary adenylate cyclase-activating peptide(1-38) (PACAP(1-38)), a 38-amino acid neuropeptide, associated with PEGylated phospholipid micelles in vivo. Both peptides were incubated for 2 h with SSM composed of PEG with molecular mass of 2000 or 5000 grafted onto distearoyl-phosphatidylethanolamine (DSPE-PEG2000 or DSPE-PEG5000) before use. We found that regardless of peptide molecular mass, PEG chain length had no significant effects on peptide-SSM interactions. Using intravital microscopy, VIP associated with DSPE-PEG5000 SSM or DSPE-PEG2000 SSM incubated at 25 degrees C evoked similar vasodilation in the intact hamster cheek pouch microcirculation. Likewise, PACAP(1-38)-induced vasodilation was PEG chain length-independent. However, SSM-associated PACAP(1-38) evoked significantly smaller vasodilation than that evoked by SSM-associated VIP (P < 0.05) at 25 degrees C. When the incubation temperature was increased to 37 degrees C, SSM-associated PACAP(1-38)-induced vasodilation was now similar to that of SSM-associated VIP. This response was associated with a corresponding increase in alpha-helix content of both peptides in the presence of phospholipids. Collectively, these data indicate that for a larger amphipathic peptide, such as PACAP(1-38), greater kinetic energy or longer incubation period is required to optimize peptide-SSM interactions and amplify peptide bioactivity in vivo.     

Molecular study on cloned endoglucanase gene from rumen bacterium

An endoglucanase gene was subcloned from anaerobic rumen bacterium Ruminococcus flavefaciens strain 17. To express endoglucanase gene in Escherichia coli and Streptococcus bovis JB1, an endoglucanase gene fragment was inserted into pVA838-based shuttle vectors. Removal of endoglucanase gene promoter and expression of endoglucanase by promoter of S. bovis JB1 alpha-amylase gene (pACMCS) was also achieved. Survival of constructs pVACMCI, pTACMC and pACMCS, which carry endoglucanase gene, and stability of endoglucanase gene in S. bovis JB1, were observed. Maximal endoglucanase activities from S. bovis JB1/pVACMCI were 2- to 3-fold higher than from E. coli/pVACMCI. Specific cell activity of E. coli/pACMCS was found to be approximately 2- to -3 fold higher than the both E. coli/pVACMCI and E. coli/pTACMC. Specific cell activity of S. bovis JB1/pACMCS was also found to be approximately 2-fold higher than the both S. bovis/pVACMCI and S. bovis JB1/pTACMC.     

Effect of stress-induced lipid peroxidation on functions of rat peritoneal macrophages

The aim of the present study was to investigate the effects of stress-induced lipid peroxidation on macrophages' functions. Animals were subjected to 4 h immobilization at 4 degrees C in restraining devices. The peritoneal macrophages obtained from rats exposed to cold and restraint stress exhibited an increase in lipid peroxidation and a decline of chemotaxis and phagocytosis compared with control rats. After supplementation with vitamin E, the increment in thiobarbituric acid reactive substances (TBARS) content as the oxidative stress marker and the decline of chemotaxis and phagocytosis in peritoneal macrophages observed during cold-restraint stress was significantly removed. No significant change in catalase activity of peritoneal macrophages was observed in groups exposed to cold-restraint stress and treated with vitamin E. These findings indicate that phagocytic and chemotactic capacities of peritoneal macrophages are decreased by cold-restraint stress and this effect of stress may be related to lipid peroxidation.     

Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein

The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.     

Quantitative microinjection of trehalose into mouse oocytes and zygotes,and its effect on development

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.     

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.     

Adenosine triphosphate alters the selenite-induced contracture and negative inotropic effect on cardiac muscle contractions

Selenium is known to play an important role in the physiology of many different cell types and extracellular application of selenite causes cellular dysfunction in many different types of tissues. In a previous study, we have shown that in rat ventricles, sodium selenite (≥1 mM) caused an increase in the resting tension and a decrease in contractile force, in a time-dependent manner. In the present study, we have shown that sodium selenite caused a contracture state both in Langendorff perfused hearts and isolated papillary muscles. We also showed that the application of extracellular ATP (0.1 mM) markedly reduced this detrimental effect of sodium selenite on ventricular contraction in Langendorff perfused hearts and delayed it in isolated papillary muscle preparations. In contrast, isoproterenol (0.1 μM) did not seem to influence this action of sodium selenite in papillary muscle preparations. Possible reasons for this protective effect of ATP to selenite-induced contracture are also discussed.