Calmodulin Inhibitors from Aspergillus stromatoides

An organic extract was prepared from the culture medium and mycelia of the marine fungus Aspergillus stromatoides Raper & Fennell . The extract was fractionated via column chromatography, and the resulting fractions were tested for their abilities to quench the fluorescence of the calmodulin (CaM) biosensor hCaM M124C‐mBBr. From the active fraction, emodin ( 1 ) and ω‐hydroxyemodin ( 2 ) were isolated as CaM inhibitors. Anthraquinones 1 and 2 quenched the fluorescence of the hCaM M124C‐mBBr biosensor in a concentration‐dependent manner with K_d values of 0.33 and 0.76 μM , respectively. The results were compared with those of chlorpromazine (CPZ), a classical inhibitor of CaM, with a K_d value of 1.25 μM . Docking analysis revealed that 1 and 2 bind to the same pocket of CPZ. The CaM inhibitor properties of 1 and 2 were correlated with some of their reported biological properties. Citrinin ( 3 ), methyl 8‐hydroxy‐6‐methyl‐9‐oxo‐9H‐xanthene‐1‐carboxylate ( 4 ), and coniochaetone A ( 5 ) were also isolated in the present study. The X‐ray structure of 5 is reported for the first time.     

Morphological changes, chitinolytic enzymes and hydrophobin-like proteins as responses of Lecanicillium lecanii during growth with hydrocarbon

Lecanicillium lecanii, Verticillium chlamydosporium, V. fungicola var flavidum and Beauveria bassiana were evaluated on their growth with pure n-hexane, toluene and n-hexane:toluene 17:83 (v:v) mixture. Another set of treatments were conducted with colloidal chitin as additional carbon source. All the strains of Lecanicillium were able to grow using hydrocarbons with or without the addition of chitin, although the presence of hydrocarbons showed significant inhibition evidenced by measured biomass, radial growth and microscopic analyses. Degradation of n-hexane ranged within 43 and 62 % and it was higher than that with toluene. The strains L460, L157 and L2149, which presented the highest growth, were further selected for determinations of hydrocarbon consumptions in microcosms. Strain L157 showed the highest consumption of n-hexane (55.6 %) and toluene (52.9 %) as sole carbon source and it also displayed activities of endochitinases, N-acetylhexosaminidase and production of hydrophobins class I and II.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Insulin-Receptor Substrate-2 (IRS-2) Is Required for Maintaining Glucokinase and Glucokinase Regulatory Protein Expression in Mouse Liver

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(−/−) mice where Irs2 was reintroduced specifically into pancreatic β-cells [RIP-Irs-2/IRS-2(−/−)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(−/−) mice. However, in RIP-Irs-2/IRS-2(−/−) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(−/−) mice. GK and GKRP mRNA levels in liver of IRS-2(−/−) were significantly lower, whereas in RIP-Irs-2/IRS-2(−/−) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(−/−) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(−/−) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the β-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(−/−) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.     

Effect of Statin Therapy in the Outcome of Bloodstream Infections Due to Staphylococcus aureus: A Prospective Cohort Study

Introduction

Statins have pleiotropic effects that could influence the prevention and outcome of some infectious diseases. There is no information about their specific effect on Staphylococcus aureus bacteremia (SAB).

Methods

A prospective cohort study including all SAB diagnosed in patients aged ≥18 years admitted to a 950-bed tertiary hospital from March 2008 to January 2011 was performed. The main outcome variable was 14-day mortality, and the secondary outcome variables were 30-day mortality, persistent bacteremia (PB) and presence of severe sepsis or septic shock at diagnosis of SAB. The effect of statin therapy at the onset of SAB was studied by multivariate logistic regression and Cox regression analysis, including a propensity score for statin therapy.

Results

We included 160 episodes. Thirty-three patients (21.3%) were receiving statins at the onset of SAB. 14-day mortality was 21.3%. After adjustment for age, Charlson index, Pitt score, adequate management, and high risk source, statin therapy had a protective effect on 14-day mortality (adjusted OR = 0.08; 95% CI: 0.01–0.66; p = 0.02), and PB (OR = 0.89; 95% CI: 0.27–1.00; p = 0.05) although the effect was not significant on 30-day mortality (OR = 0.35; 95% CI: 0.10–1.23; p = 0.10) or presentation with severe sepsis or septic shock (adjusted OR = 0.89; CI 95%: 0.27–2.94; p = 0.8). An effect on 30-day mortality could neither be demonstrated on Cox analysis (adjusted HR = 0.5; 95% CI: 0.19–1.29; p = 0.15).

Conclusions

Statin treatment in patients with SAB was associated with lower early mortality and PB. Randomized studies are necessary to identify the role of statins in the treatment of patients with SAB.     

From START to FINISH: The Influence of Osmotic Stress on the Cell Cycle

The cell cycle is a sequence of biochemical events that are controlled by complex but robust molecular machinery. This enables cells to achieve accurate self-reproduction under a broad range of different conditions. Environmental changes are transmitted by molecular signalling networks, which coordinate their action with the cell cycle. The cell cycle process and its responses to environmental stresses arise from intertwined nonlinear interactions among large numbers of simpler components. Yet, understanding of how these pieces fit together into a coherent whole requires a systems biology approach. Here, we present a novel mathematical model that describes the influence of osmotic stress on the entire cell cycle of S. cerevisiae for the first time. Our model incorporates all recently known and several proposed interactions between the osmotic stress response pathway and the cell cycle. This model unveils the mechanisms that emerge as a consequence of the interaction between the cell cycle and stress response networks. Furthermore, it characterises the role of individual components. Moreover, it predicts different phenotypical responses for cells depending on the phase of cells at the onset of the stress. The key predictions of the model are: (i) exposure of cells to osmotic stress during the late S and the early G2/M phase can induce DNA re-replication before cell division occurs, (ii) cells stressed at the late G2/M phase display accelerated exit from mitosis and arrest in the next cell cycle, (iii) osmotic stress delays the G1-to-S and G2-to-M transitions in a dose dependent manner, whereas it accelerates the M-to-G1 transition independently of the stress dose and (iv) the Hog MAPK network compensates the role of the MEN network during cell division of MEN mutant cells. These model predictions are supported by independent experiments in S. cerevisiae and, moreover, have recently been observed in other eukaryotes.     

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals

Background

Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.

Methods

We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.

Results

The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “full-responders” (positive response against both viral particles), “partial-responders” (positive response only against NL4-3/ΔRT virions) and “non-responders” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.

Conclusions

In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay.