Biochemical characterization of RssA-RssB, a two-component signal transduction system regulating swarming behavior in Serratia marcescens

Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37 degrees C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30 degrees C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfF(Sm)), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.     

Chaperone-mediated folding and maturation of the penicillin acylase precursor in the cytoplasm of Escherichia coli

Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The outcome of proPAC folding and PAC maturation could be affected by several factors, such as inducer type, proPAC formation rate, and chaperone availability. Misfolding of proPAC in the cytoplasm could be partially resolved through the coexpression of cytoplasmic chaperones, such as trigger factor, GroEL/ES, or DnaK/J-GrpE. The three chaperones tested showed different extents of the effect on proPAC solublization and PAC maturation, and trigger factor had the most prominent one. However, the chaperone-mediated solublization of proPAC did not guarantee its maturation, which is usually limited by the first autoproteolytic step. It was observed that arabinose could act as an effective inducer for the induction of LL pac expression regulated by the lac-derived promoter system of trc. In addition, PAC maturation could be highly facilitated by arabinose supplementation and coexpression of trigger factor, suggesting that the coordination of chaperone systems with proper culture conditions could dramatically impact recombinant protein production. This study suggests that folding/misfolding of proPAC could be a major step limiting the overproduction of PAC in E. coli and that the problem could be resolved through the search for appropriate chaperones for coexpression. It also demonstrates the analogy in the issues of proPAC misfolding as well as the expression bottleneck occurring in the cytoplasm (i.e., LL pac expression) and those occurring in the periplasm (i.e., wild-type pac expression).     

Brainstem Neurons Survive the Identical Ischemic Stress That Kills Higher Neurons: Insight to the Persistent Vegetative State

Global ischemia caused by heart attack, pulmonary failure, near-drowning or traumatic brain injury often damages the higher brain but not the brainstem, leading to a ‘persistent vegetative state’ where the patient is awake but not aware. Approximately 30,000 U.S. patients are held captive in this condition but not a single research study has addressed how the lower brain is preferentially protected in these people. In the higher brain, ischemia elicits a profound anoxic depolarization (AD) causing neuronal dysfunction and vasoconstriction within minutes. Might brainstem nuclei generate less damaging AD and so be more resilient? Here we compared resistance to acute injury induced from simulated ischemia by ‘higher’ hippocampal and striatal neurons versus brainstem neurons in live slices from rat and mouse. Light transmittance (LT) imaging in response to 10 minutes of oxygen/glucose deprivation (OGD) revealed immediate and acutely damaging AD propagating through gray matter of neocortex, hippocampus, striatum, thalamus and cerebellar cortex. In adjacent brainstem nuclei, OGD-evoked AD caused little tissue injury. Whole-cell patch recordings from hippocampal and striatal neurons under OGD revealed sudden membrane potential loss that did not recover. In contrast brainstem neurons from locus ceruleus and mesencephalic nucleus as well as from sensory and motor nuclei only slowly depolarized and then repolarized post-OGD. Two-photon microscopy confirmed non-recoverable swelling and dendritic beading of hippocampal neurons during OGD, while mesencephalic neurons in midbrain appeared uninjured. All of the above responses were mimicked by bath exposure to 100 µM ouabain which inhibits the Na⁺/K⁺ pump or to 1–10 nM palytoxin which converts the pump into an open cationic channel.Therefore during ischemia the Na⁺/K⁺ pump of higher neurons fails quickly and extensively compared to naturally resilient hypothalamic and brainstem neurons. The selective survival of lower brain regions that maintain vital functions will support the persistent vegetative state.     

Modelling the effect of treatment and behavioral change in HIV transmission dynamics

In this paper we analyze a model for the HIV-infection transmission in a male homosexual population. In the model we consider two types of infected individuals. Those that are infected but do not know their serological status and/or are not under any sort of clinical /therapeutical treatment, and those who are. The two groups of infectives differ in their incubation time, contact rate with susceptible individuals, and probability of disease transmission. The aim of this article is to study the roles played by detection and changes in sexual behavior in the incidence and prevalence of HIV. The analytical results show that there exists a unique endemic equilibrium which is globally asymptotically stable under a range of parameter values whenever a detection /treatment rate and an indirect measure of the level of infection risk are sufficiently large. However, any level of detection/ treatment rate coupled with a decrease of the transmission probability lowers the incidence rate and prevalence level in the population. In general, only significant reductions in the transmission probability (achieved through, for example, the adoption of safe sexual practices) can contain effectively the spread of the disease.     

Kinetics of T helper subsets and associated cytokines correlate well with the clinical activity of graft-versus-host disease

Background

CD4⁺interferon (IFN)-γ⁺ T cell (Th1) and CD4⁺interleukin (IL)-4⁺ T cell (Th2) polarizations are crucial in the pathogenesis of graft-versus-host disease (GVHD). However, this hypothesis is largely based on animal experiments of Parent-into-F1 GVHD model. The causal relationship between kinetics of Th1, Th2 and associated cytokines and the clinical activity of GVHD in a real world situation remains unknown.

Methodology

Peripheral blood was collected every week prospectively from Day 0 to Day 210 (patients without GVHD) or Day 300 (patients with chronic GVHD) after allogeneic peripheral blood stem cell transplantation in consecutive 27 patients. The frequencies of Th1 and Th2 within CD4⁺ T cells were determined by flow cytometry and pplasma IFN-γ, IL-12, IL-4, and IL-10 were determined by ELISA.

Principal Findings

Kinetics of Th1, Th2 frequency, and the plasma IL-10 and IFN-γ more commonly coincided with, rather than predicted, the activity of GVHD. These markers are significantly higher when acute or chronic GVHD developed. The kinetics of IL-10 is especially correlated well with the activity of GVHD during clinical course of immunosuppressive treatment. For patients with hepatic GVHD, there is a positive correlation between plasma IL-10 levels and the severity of hepatic injury. The frequency of Th2 is also significant higher in acute GVHD and tends to be higher in chronic GVHD. Interestingly, there is a very good positive correlation between the frequency of Th1 and Th2 (r = 0.951, p<0.001). The plasma level of IL-4 and IL-12 are not associated with the activity of GVHD.

Conclusions

The frequency of Th1, Th2 within CD4⁺ T cells and plasma IL-10 and IFN-γ are good biomarkers of GVHD. Plasma IL-10 can also be used to monitor the therapeutic responsiveness. Furthermore, both Th1 and Th2 likely contribute to the pathogenesis of GVHD.     

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence

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Taurine resumed neuronal differentiation in arsenite-treated N2a cells through reducing oxidative stress,endoplasmic reticulum stress,and mitochondrial dysfunction

The goal of the study is to investigate the preventive effect of taurine against arsenite-induced arrest of neuronal differentiation in N2a cells. Our results revealed that taurine reinstated the neurite outgrowth in arsenite-treated N2a cells. Meanwhile, arsenite-induced oxidative stress and mitochondrial dysfunction as well as degradation of mitochondria DNA (mtDNA) were also inhibited by co-treatment of taurine. Since oxidative stress and mitochondrial dysfunction is closely associated with endoplasmic reticulum (ER) stress, we further examined indicators of ER stress, 78 kDa glucose-regulated protein (GRP78), and C/EBP-homologous protein (CHOP) protein expression. The results demonstrated that taurine significantly reduced arsenite-induced ER stress in N2a cells. In the parallel experiment, arsenite-induced disruption of intracellular calcium homeostasis was also ameliorated by taurine. The proven bio-function of taurine preserved a preventive effect against deleteriously cross-talking between oxidative stress, mitochondria, and ER. Overall, the results of the study suggested that taurine reinstated neuronal differentiation by inhibiting oxidative stress, ER stress, and mitochondrial dysfunction in arsenite-treated N2a cells.