Seasonal variations of phage life strategies and bacterial physiological states in three northern temperate lakes

The current consensus concerning the prevalence of lytic and lysogenic phage life cycles in aquatic systems is that the host physiological state may influence viral strategies, lysogeny being favoured when hosts have reduced metabolic rates. We explored this hypothesis, by following phage cycle dynamics, host physiological state and metabolic activity over an annual cycle in three lakes subjected to strong seasonal fluctuations, including 4–5 months of ice cover. We observed marked seasonal dynamics of viral and bacterial communities, with low bulk and cell‐specific bacterial metabolism in winter, and a dramatic increase in injured bacteria under the ice cover in all lakes. This period was accompanied by contrasting patterns in the proportion of lysogenic cells. In the eutrophic lake, times of low bacterial metabolic rates and high proportion of damaged cells corresponded to highest levels of lysogeny, supporting the notion that hosts are a ‘refuge’ for viruses. In the two unproductive lakes, peaks of injured cells corresponded to a minimum of lysogeny, suggesting an ‘abandon the sinking ship’ response, where the prophage replicates before the loss of genome. We suggest that these diverging responses to the host physiological state are not contradictory, but rather that there may be thresholds of cell stress and metabolic activity leading to one or the other response.     

A Novel Mouse Model for Stable Engraftment of a Human Immune System and Human Hepatocytes

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2^-/- IL-2Rγc^-/- NOD.sirpa uPA^tg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20–50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.     

Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel which plays a major role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly known. RINm5F cells who express almost exclusively (approximately 90%) the IP3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP3R-3-mediated Ca2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca2+ signalling cascade, whereby activated PKC dampens IP3R-3 activity.     

Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP₃R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺ concentration. There are three subtypes of IP₃R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP₃R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP₃R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP₃R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP₃R‐3 in intact RINm5F cells. [³H]IP₃ (inositol 1,4,5‐trisphosphate) binding affinity and IP₃‐induced Ca²⁺ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP₃R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca²⁺ signalling systems act co‐operatively in endocrine cell responses to external stimuli.     

Regulation of blood volume during weightlessness simulation of long duration

To study the effects of microgravity on the mechanisms involved in the regulation of body hydrous status, total body water (TBW), plasma volume (PV), and its main regulating hormones (plasma renin, aldosterone, atrial natriuretic peptide (ANP), anti-diuretic hormone (ADH)) were determined, by isotopic dilution, Dill and Costill's formula, and radio-immunologic dosages, in 9 male subjects submitted to a 90-d head-down bed rest (HDBR). ADH was determined in 24 h urinary collection as well as osmolality, sodium, and potassium. Body mass decreased (-2.8 +/- 0.8 kg) as well as TBW(-7.2% +/- 0.9%, i.e., -2.6 +/- 0.7 kg) and PV (-4.7% +/- 1.8%). Renin and aldosterone were enhanced (+109.0% +/- 15.4% and +87.2% +/- 38.9%, respectively). Simultaneously, we observed a decrease in ANP (-33.2% +/- 20.4%). Other variables, including ADH, were not affected by HDBR. Body mass and TBW decrease (and consequently lean body mass) are associated with muscle atrophy. Renin, aldostrerone, and ANP modifications are well explained by the decrease in PV, which was not enough to induce ADH changes. It suggests that in man, the main regulatory factor for ADH secretion is osmolality, when PV is modestly and progressively decreased without arterial pressure modification, which was the case in the present protocol.     

Analysis of slow-binding enzyme inhibitors at elevated enzyme concentrations

The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant that is much lower than the initial dissociation constant with enzyme is discussed. For these systems, inhibition is rapid and low steady-state product concentrations are produced at saturating inhibitor concentrations. By working at elevated enzyme concentrations, improved signal-to-noise ratios are achieved and data may be collected at saturating inhibitor levels. Numerical simulations confirmed that improved parameter estimates are obtained and useful data to discern the mechanism of slow-binding inhibition are produced by working at elevated enzyme concentrations. The saturation kinetics that were unobservable in two previous studies of an enzyme inhibitor system were measured by performing experiments at an elevated enzyme concentration. These results indicate that consideration of the quality of the data acquired using a particular assay is an important factor when selecting the enzyme concentration at which to perform experiments used to characterize the class of enzyme inhibitors examined herein.     

Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.     

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.