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.     

Evidence That Mast Cells Are Not Required for Healing of Splinted Cutaneous Excisional Wounds in Mice

Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-Kit^W-sh/W-sh, WBB6F₁-Kit^W/W-v, and Cpa3-Cre; Mcl-1^fl/fl mice re-epithelialized splinted excisional skin wounds at rates very similar to those in the corresponding wild type or control mice. Furthermore, at the time of closure, scars were similar in the genetically mast cell-deficient mice and the corresponding wild type or control mice in both quantity of collagen deposition and maturity of collagen fibers, as evaluated by Masson’s Trichrome and Picro-Sirius red staining. These data indicate that mast cells do not play a significant non-redundant role in these features of the healing of splinted full thickness excisional cutaneous wounds in mice.     

The Functional Significance of MicroRNA-29c in Patients with Colorectal Cancer: A Potential Circulating Biomarker for Predicting Early Relapse

Background

The recurrence of colorectal cancer (CRC) is frequent within the first year of curative resection surgery and may be unavoidable. microRNAs have been suggested to play roles in carcinogenesis and cancer recurrence. We recently identified microRNA-29c (miRNA-29c) as a predictor of early recurrence in CRC. In the present study, we further investigated the functions and serum level of miRNA-29c in relation to early recurrence of CRC.

Methods

First we further confirmed overexpression of miRNA-29c in non-early relapse subjects. Gain-of-function in vitro studies were used to evaluate the effect of miRNA-29c on cell proliferation, migration, invasion, and cell cycle progression. The colon cancer cell line Caco2 and a stable clone overexpressing miRNA-29c were xenografted to evaluate the in vivo effect of miRNA-29c in null mice. Finally, circulating miRNA-29c was investigated as a potential biomarker for identifying early relapse.

Results

miRNA-29c expression significantly decreased during early relapse compared to non-early relapse in UICC stage II and III CRC patients (P = 0.021). In vitro studies showed that overexpression of miRNA-29c inhibited cell proliferation and migration. The cell cycle studies also revealed that miRNA-29c caused an accumulation of the G1 and G2 population. In vivo, miRNA-29c suppressed tumor growth in null mice. The serum miRNA-29c increased significantly in early relapsed patients compared to non-early elapsed patients (P = 0.012).

Conclusions

miRNA-29c shows anti-tumorigenesis activity, and preoperative circulating miRNA-29c levels can be used to predict postoperative early relapse of CRC.     

Histone Deacetylase Inhibitor Romidepsin Induces HIV Expression in CD4 T Cells from Patients on Suppressive Antiretroviral Therapy at Concentrations Achieved by Clinical Dosing

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC₅₀ = 4.5 nM) compared with vorinostat (VOR; EC₅₀ = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC₅₀ = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.     

Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.     

A common allergenic epitope of Bermuda grass pollen shared by other grass pollens

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9–13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9–13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29–36 kD. The cross-reactivity between BGP andLol pI, the group I allergen of rye grass pollen, was further evaluated;Lol pI was recognized by MAb 9–13, but not by our MAbs/polyclonal antiserum againstCyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9–13 is a common (C) epitope shared byLol pI andCyn d Bd68K, 48K, 38K, andCyn dI does not share significant antigenicity withLol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9–13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction.     

Determination of urinary beryllium,arsenic, and selenium in steel production workers

Some of the most pernicious dangers of pollution arise from the presence of traces of toxic elements in the environment. In this work, we report on the determination of beryllium, arsenic, and selenium in the urine of steel production and steel quality control (QC) workers, in comparison to healthy control subjects. The urine samples were digested by a microwave system. Graphite furnace and hydride atomic absorption was used for the quantitative measurements of Be and As and Se, respectively. A quality control method for these procedures was established with concurrent analysis of Standard Trace Metals 7879 Level II and NIST SRM 2670 (Toxic Elements in Freeze Dried Urine). The results show that the urinary levels of these elements in steel production (As, 38.1±28.7 μg/L; Be, 1.58±0.46 μg/L, and Se, 69.2±28.8μg/L) and in quality control workers (As, 23.9±18.1 μg/L; Be, 1.58±0.46 μg/L, and Se, 54.8±25.1 μg/L) are significantly higher than in the controls (As, 10.3±8.7 μg/L; Be, 0.83±0.46 μg/L; and Se, 32.3±13.5 μg/L). The possible connection of these elements with the etiology of disease and the possible role of selenium as a protective agent against the oncogenic and teratogenic action of other substances is discussed. We suggest the need for improvement of environmental conditions in the workplace through better ventilation and industrial hygiene practices.     

The differential effects of tamoxifen and ICI 182,780 on the reduction of Na+/K+ ATPase activity and spontaneous oscillations by 17beta-estradiol

A prolonged treatment with 17beta-estradiol reduces the frequency of spontaneous oscillations and the Na+/K+ ATPase activity in rat uteri. Acute inhibition of Na+/K+ ATPase activity by a Na+/K+ ATPase inhibitor, ouabain, decreases the frequency of oxytocin-induced oscillations in uteri. Therefore, the purpose of this study was to examine whether the prolonged inhibition of Na+/K+ ATPase activity by 17beta-estradiol was estrogen receptor (ER)-dependent. The uterine explants from ovariectomized rats were cultured in vitro as our experimental model to compare the effect of two antiestrogenic compounds (ICI 182,780 and tamoxifen) on the Na+/K+ ATPase activity and the frequency of spontaneous oscillations. ATPase assay and a standard muscle bath apparatus were to measure the activity and the contraction. When compared with the control, a 2-day treatment with 17beta-estradiol in vivo or in vitro decreased the activity and the frequency. ICI 182,780 lowered the activity but tamoxifen did not. ICI 182,780 did not decrease the frequency but tamoxifen did. Even the reversal effects of these antiestrogenic compounds on the reduced activity and the frequency by 17beta-estradiol were different. Tamoxifen elicited a greater reversal effect on the reduced activity but ICI 182,780 did not. In contrast, ICI 182,780 elicited a greater reversal effect on the reduced frequency but tamoxifen did not. Prolonged inhibition of Na+/K+ ATPase activity by K+-free solution suppressed the frequency with the elevation of basal tension. Addition of KCl at lower concentrations (0.3-1.2 mM) induced oscillatory contraction after reducing the basal tension. As our data suggest, the prolonged effect of 17beta-estradiol may decrease uterine the activity through ER dependent and independent pathways. The reduction of uterine Na+/K+ ATPase activity by estrogens may increase the basal tension after each oscillatory cycle, which, in part, contributes to the reduced frequency of spontaneous oscillations.     

Assessing the complex sponge microbiota: core,variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world''s oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.     

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.