Characterization of the dimerization process of a domain-swapped dimeric variant of human pancreatic ribonuclease

It has been previously reported that the structure of a human pancreatic ribonuclease variant, namely PM8, constitutes a dimer by the exchange of an N-terminal domain, although in an aqueous solution it is found mainly as a monomer. First, we investigated the solution conditions that favour the dimerization of this variant. At 29 degrees C in a 20% (v/v) ethanol buffer, a significant fraction of the protein is found in dimeric form without the appearance of higher oligomers. This dimer was isolated by size-exclusion chromatography and the dimerization process was studied. The dissociation constant of this dimeric form is 5 mm at 29 degrees C. Analysis of the dependence of the dimerization process on the temperature shows that unlike bovine pancreatic ribonuclease, a decrease in the temperature shifts the monomer-dimer equilibrium to the latter form. We also show that a previous dissociation of the exchangeable domain from the main protein body does not take place before the dimerization process. Our results suggest a model for the dimerization of PM8 that is different to that postulated for the dimerization of the homologous bovine pancreatic ribonuclease. In this model, an open interface is formed first and then intersubunit interactions stabilize the hinge loop in a conformation that completely displaces the equilibrium between nonswapped and swapped dimers to the latter one.     

Effect of ice melting on bacterial carbon fluxes channelled by viruses and protists in the Arctic Ocean

During the last few years, extensive sea ice melting in the Arctic due to climate change has been detected, which could potentially modify the organic carbon fluxes in these waters. In this study, the effect of sea ice melting on bacterial carbon channelling by phages and protists has been evaluated in the northern Greenland Sea and Arctic Ocean. Grazing on bacteria by protists was evaluated using the FLB disappearance method. Lysis of bacteria due to viral infections was measured using the virus reduction approach. Losses of bacterial production caused by protists (PMM_BP) dominated losses caused by viruses (VMM_BP) throughout the study. Lysogenic viral production was detected in 7 out of 21 measurements and constituted from 33.9 to 100.0% of the total viral production. Significantly higher PMM_BP and lower VMM_BP were detected in waters affected by ice melting compared with unaffected waters. Consequently, significantly more bacterial carbon was channelled to the higher trophic levels in affected waters (13.05 ± 5.98 μgC l⁻¹ day⁻¹) than in unaffected waters (8.91 ± 8.33 μgC l⁻¹ day⁻¹). Viruses channelled 2.63 ± 2.45 μgC l⁻¹ day⁻¹ in affected waters and 4.27 ± 5.54 μgC l⁻¹ day⁻¹ in unaffected waters. We conclude that sea ice melting in the Arctic could modify the carbon flow through the microbial food web. This process may be especially important in the case of massive sea ice melting due to climate change.     

Phenotypic and genotypic characterization of rhizobia associated with Acacia saligna (Labill.) Wendl. in nurseries from Algeria

Twenty seven rhizobial strains associated with Acacia saligna grown in northern and southern Algeria were characterized, including generation time, host-range, the 16S rRNA gene and 16S–23S rRNA intergenic spacer restriction patterns, 16S rRNA gene sequence analysis and tolerance to salinity and drought. Cross inoculation tests indicated that 11 slow-growing isolates from northern nurseries were able to nodulate introduced Australian acacias exclusively, whereas 16 fast-growing isolates, mainly from southern nurseries, were capable of also nodulating native acacias. Restriction patterns and sequence analysis of the 16S rRNA gene showed that strains of the first group belonged to Bradyrhizobium while strains of the second group were related to Sinorhizobium meliloti and Rhizobium gallicum. Interestingly, five strains of the first group formed a distinct cluster phylogenetically close to Bradyrhizobium betae, a non-nodulating species causing tumour-like deformations in sugar beet roots. Bradyrhizobium strains were in general more sensitive to NaCl and PEG than the S. meliloti and R. gallicum representatives. Among the latter, strains S. meliloti BEC1 and R. gallicum DJA2 were able to tolerate up to 1 M NaCl and 20% PEG. This, together with their wide host-range among Acacia species, make them good candidates for developing inoculants for A. saligna and other acacia trees growing in arid areas.     

Paternal levels of DNA damage in spermatozoa and maternal parity influence offspring mortality in an endangered ungulate

Understanding which factors influence offspring mortality rates is a major challenge since it influences population dynamics and may constrain the chances of recovery among endangered species. Most studies have focused on the effects of maternal and environmental factors, but little is known about paternal factors. Among most polygynous mammals, males only contribute the haploid genome to their offspring, but the possibility that sperm DNA integrity may influence offspring survival has not been explored. We examined several maternal, paternal and individual factors that may influence offspring survival in an endangered species (Gazella cuvieri). Levels of sperm DNA damage had the largest impact upon offspring mortality rates, followed by maternal parity. In addition, there was a significant interaction between these two variables, so that offspring born to primiparous mothers were more likely to die if their father had high levels of sperm DNA damage, but this was not the case among multiparous mothers. Thus, multiparous mothers seem to protect their offspring from the deleterious effects of sperm DNA damage. Since levels of sperm DNA damage seem to be higher among endangered species, more attention should be paid to the impact of this largely ignored factor among the viability of endangered species.     

Phytosterol plasma concentrations and coronary heart disease in the prospective Spanish EPIC cohort

Phytosterol intake with natural foods, a measure of healthy dietary choices, increases plasma levels, but increased plasma phytosterols are believed to be a coronary heart disease (CHD) risk factor. To address this paradox, we evaluated baseline risk factors, phytosterol intake, and plasma noncholesterol sterol levels in participants of a case control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) Spanish cohort who developed CHD (n = 299) and matched controls (n = 584) who remained free of CHD after a 10 year follow-up. Sitosterol-to-cholesterol ratios increased across tertiles of phytosterol intake (P = 0.026). HDL-cholesterol level increased, and adiposity measures, cholesterol/HDL ratios, and levels of glucose, triglycerides, and lathosterol, a cholesterol synthesis marker, decreased across plasma sitosterol tertiles (P < 0.02; all). Compared with controls, cases had nonsignificantly lower median levels of phytosterol intake and plasma sitosterol. The multivariable-adjusted odds ratio for CHD across the lowest to highest plasma sitosterol tertile was 0.59 (95% confidence interval, 0.36–0.97). Associations were weaker for plasma campesterol. The apolipoprotein E genotype was unrelated to CHD risk or plasma phytosterols. The data suggest that plasma sitosterol levels are associated with a lower CHD risk while being markers of a lower cardiometabolic risk in the EPIC-Spain cohort, a population with a high phytosterol intake.     

Positive selection versus demography: evolutionary inferences based on an unusual haplotype structure in Drosophila simulans

Coalescent simulations were used to investigate the possible role of population subdivision and history in shaping nucleotide variation in a recombining 88-kb genomic fragment of Drosophila simulans displaying an unusual large-scale haplotype structure. The multilocus analysis, based on summary statistics using specific demographic null models under recombination, indicates that the observed levels of linkage disequilibrium differed significantly from the values expected under different bottleneck and population admixture scenarios. These results indicate that demography alone may not account for the observed pattern of variation and support the previous claim that the data are better described by a model in which an adaptive mutation has not yet gone to fixation.     

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.     

Norepinephrine induces calcium spikes and proinflammatory actions in human hepatic stellate cells

Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through alpha1-adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigated the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory, and fibrogenic actions in the injured liver. Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was studied in fura-2-loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC II) phosphorylation. Cell proliferation and collagen-alpha1(I) expression were assessed by [3H]thymidine incorporation and quantitative PCR, respectively. NF-kappaB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Normal human livers expressed alpha(1A)-adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed alpha(1A)-adrenoceptors. NE induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes). Prazosin (alpha1-blocker) completely prevented NE-induced Ca2+ spikes, whereas propranolol (nonspecific beta-blocker) partially attenuated this effect. NE caused phosphorylation of MLC II and cell contraction. In contrast, NE did not affect cell proliferation or collagen-alpha1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. NE stimulated NF-kappaB activation. BAY 11-7082, a specific NF-kappaB inhibitor, blocked NE-induced chemokine secretion. We conclude that NE stimulates NF-kappaB and induces cell contraction and proinflammatory effects in human HSC. Catecholamines may participate in the pathogenesis of portal hypertension and liver fibrosis by targeting HSC.