Mode of action investigation for the antibacterial cationic anthraquinone analogs

Reported previously by our group, we have developed a novel class of antibacterial cationic anthraquinone analogs with superb potency (MIC <1μg/mL) against Gram positive (G+) pathogens including Methicillin-resistant Staphylococcus aureus (MRSA). However, most of these compounds only manifest modest antibacterial activity against Gram negative (G-) bacteria. Further investigation on the antibacterial mode of action using fluorogenic dyes reveals that these compounds exert two different modes of action that account for the difference in their antibacterial profile. It was found that most of the compounds exert their antibacterial activity by disrupting the redox processes of bacteria. At high concentration, these compounds can also act as membrane disrupting agents. This information can help to design new therapeutics against various bacteria.     

Limited gene flow may enhance adaptation to local optima in isolated populations of the Roesel's bush cricket (Metrioptera roeselii)

Variation in morphological traits along latitudinal gradients often manifests as size clines. In insects, both positive and negative correlations are seen, and the mechanism behind the response is unclear. We studied variation in seven morphological traits of Roesel's bush cricket, Metrioptera roeselii, sampled from seven latitude-matched-pair populations that were either geographically isolated from or connected to the species continuous distribution range. The aim was to examine whether morphological traits differed between isolated and continuous populations, and whether latitudinal variation was apparent. The data were used to indicate whether variation in trait means originates from plastic responses to the environment or genetic adaptation to local conditions. To evaluate the influence of gene flow on trait means, we analysed the genetic variation in seven microsatellites. Data showed that individuals from isolated populations display a positive relationship between latitude and body size, whereas individuals from continuous populations show little or no such relationship. The combined morphological and genetic data suggest that the isolated populations have adapted to local optima, while gene flow between continuous populations appears to counteract this process.     

Ubiquitylation and developmental regulation of invariant surface protein expression in trypanosomes

The cell surface of Trypanosoma brucei is dominated by the glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG), which is essential for immune evasion. VSG biosynthesis, trafficking, and turnover are well documented, but trans-membrane domain (TMD) proteins, including the invariant surface glycoproteins (ISGs), are less well characterized. Internalization and degradation of ISG65 depend on ubiquitylation of conserved cytoplasmic lysines. Using epitope-tagged ISG75 and reporter chimeric proteins bearing the cytoplasmic and trans-membrane regions of ISG75, together with multiple mutants with lysine-to-arginine mutations, we demonstrate that the cytoplasmic tail of ISG75 is both sufficient and necessary for endosomal targeting and degradation. The ISG75 chimeric reporter protein localized to endocytic organelles, while lysine-null versions were significantly stabilized at the cell surface. Importantly, ISG75 cytoplasmic lysines are modified by extensive oligoubiquitin chains and ubiquitylation is abolished in the lysine-null version. Furthermore, we find evidence for differential modes of turnover of ISG65 and ISG75. Full-length lysine-null ISG65 localization and protein turnover are significantly perturbed, but ISG75 localization and protein turnover are not, while ubiquitin conjugates can be detected for full-length lysine-null ISG75 but not ISG65. We find that the ISG75 ectodomain has a predicted coiled-coil, suggesting that ISG75 could be part of a complex, while ISG65 behaves independently. We also demonstrate a developmental stage-specific mechanism for exclusion of surface ISG expression in insect-stage cells by a ubiquitin-independent mechanism. We suggest that ubiquitylation may be a general mechanism for regulating trans-membrane domain surface proteins in trypanosomes.     

ATP and its N<Superscript>6</Superscript>-substituted analogues: parameterization,molecular dynamics simulation and conformational analysis

In this work we used a combination of classical molecular dynamics and simulated annealing techniques to shed more light on the conformational flexibility of 12 adenosine triphosphate (ATP) analogues in a water environment. We present simulations in AMBER force field for ATP and 12 published analogues [Shah et al. (1997) Proc Natl Acad Sci USA 94: 3565–3570]. The calculations were carried out using the generalized Born (GB) solvation model in the presence of the cation Mg²⁺. The ion was placed at a close distance (2 Å) from the charged oxygen atoms of the beta and gamma phosphate groups of the ?3 negatively charged ATP analogue molecules. Analysis of the results revealed the distribution of inter-proton distances H8–H1′ and H8–H2′ versus the torsion angle ψ (C4–N9-C1′–O4′) for all conformations of ATP analogues. There are two gaps in the distribution of torsion angle ψ values: the first is between ?30 and 30 degrees and is described by cis-conformation; and the second is between 90 and 175 degrees, which mostly covers a region of anti conformation. Our results compare favorably with results obtained in experimental assays [Jiang and Mao (2002) Polyhedron 21:435–438].

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Figure Dihedral O4′–C1′–N9–C4 angle dependence on inter-proton distances H8–H1′ (crosses) and H8–H2′ (dots) measured for ATP

     

Microbial community analysis and identification of alternative host-specific fecal indicators in fecal and river water samples using pyrosequencing

It is important to know the comprehensive microbial communities of fecal pollution sources and receiving water bodies for microbial source tracking. Pyrosequencing targeting the V1-V3 hypervariable regions of the 16S rRNA gene was used to investigate the characteristics of bacterial and Bacteroidales communities in major fecal sources and river waters. Diversity analysis indicated that cow feces had the highest diversities in the bacterial and Bacteroidales group followed by the pig sample, with human feces having the lowest value. The Bacteroidales, one of the potential fecal indicators, totally dominated in the fecal samples accounting for 31%-52% of bacterial sequences, but much less (0.6%) in the river water. Clustering and Venn diagram analyses showed that the human sample had a greater similarity to the pig sample in the bacterial and Bacteroidales communities than to samples from other hosts. Traditional fecal indicators, i.e., Escherichia coli, were detected in the human and river water samples at very low rates and Clostridium perfringens and enterococci were not detected in any samples. Besides the Bacteroidales group, some microorganisms detected in the specific hosts, i.e., Parasutterella excrementihominis, Veillonella sp., Dialister invisus, Megamonas funiformis, and Ruminococcus lactaris for the human and Lactobacillus amylovorus and Atopostipes sp. for the pig, could be used as potential host-specific fecal indicators. These microorganisms could be used as multiple fecal indicators that are not dependent on the absence or presence of a single indicator. Monitoring for multiple indicators that are highly abundant and host-specific would greatly enhance the effectiveness of fecal pollution source tracking.     

Cleavage of von Willebrand factor by granzyme M destroys its factor VIII binding capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF.     

Experimental and theoretical study on high temperature induced changes in chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence oscillations in barley leaves upon 2 % CO<Subscript>2</Subscript>

Oscillations in many of photosynthetic quantities with a period of about 1 min can be routinely measured with higher plant leaves after perturbation of the steady state by sudden change in gas phase. Among all hypotheses suggested so far to explain the oscillations, an effect of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activation status to control the oscillations is highly probable, at least upon high temperature (HT) treatment when in vivo RuBPCO activity controlled by RuBPCO activase (RuBPCO-A) decreases. Therefore, we measured the oscillations in fluorescence signal coming from barley leaves (Hordeum vulgare L. cv. Akcent) after their exposure for various time intervals to different HTs in darkness. We also evaluated steady state fluorescence and CO₂ exchange parameters to have an insight to functions of electron transport chain within thylakoid membrane and Calvin cycle before initiation of the oscillations. The changes in period of the oscillations induced by moderate HT (up to 43 °C) best correlated with changes in non-photochemical fluorescence quenching (q_N) that in turn correlated with changes in gross photosynthetic rate (P _G) and rate of RuBPCO activation (k_act). Therefore, we suggest that changes in period of the oscillations caused by moderate HT are mainly controlled by RuBPCO activation status. For more severe HT (45 °C), the oscillations disappeared which was probably caused by an insufficient formation of NADPH by electron transport chain within thylakoid membrane as judged from a decrease in photochemical fluorescence quenching (q_P). Suggestions made on the basis of experimental data were verified by theoretical simulations of the oscillations based on a model of Calvin cycle and by means of a control analysis of the model.     

Tumor necrosis factor-alpha stimulates gastric epithelial cell proliferation

TNF-alpha is a cytokine produced during gastric mucosal injury. We examined whether TNF-alpha could promote mucosal repair by stimulation of epithelial cell proliferation and explored further the underlying mechanisms in a rat gastric mucosal epithelial cell line (RGM-1). TNF-alpha treatment (1-10 ng/ml) for 12 or 24 h significantly increased cell proliferation but did not induce apoptosis in RGM-1 cells. TNF-alpha treatment significantly increased cytosolic phospholipase A(2) and cyclooxygenase-2 (COX-2) protein expression and PGE(2) level but did not affect the protein levels of EGF, basic fibroblast growth factor, and COX-1 in RGM-1 cells. The mRNA of TNF receptor (TNF-R) 2 but not of TNF-R1 was also increased. Dexamethasone dose dependently inhibited the stimulatory effect of TNF-alpha on cell proliferation, which was associated with a significant decrease in cellular COX-2 expression and PGE(2) level. A selective COX-2 inhibitor 3-(3-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-5,5-dimethyl-(5)H-furan-2-one (DFU) by itself had no effect on basal cell proliferation but significantly reduced the stimulatory effect of TNF-alpha on RMG-1 cells. Combination of dexamethasone and DFU did not produce an additive effect. PGE(2) significantly reversed the depressive action of dexamethasone on cell proliferation. These results suggest that TNF-alpha plays a regulatory role in epithelial cell repair in the gastric mucosa via the TNF-alpha receptor and activation of the arachidonic acid/PG pathway.     

A Study of Glutathione <Emphasis Type="Italic">S</Emphasis>-transferase pi Expression in Central Nervous System of Subjects with Amyotrophic Lateral Sclerosis Using RNA Extraction from Formalin-Fixed,Paraffin-Embedded Material

The expression of glutathione S-transferase pi (GST pi), an enzyme responsible for inactivation of a large variety of toxic compounds was studied in spinal cord, motor and sensory brain cortex obtained from patients who died in the course of amyotrophic lateral sclerosis (ALS). The studies were performed on formalin-fixed, paraffin-embedded (FFPE) and freshly frozen tissues. The method of RNA isolation from FFPE was modified. A significant decrease of GST pi-mRNA expression was found in cervical spinal cord and motor brain cortex of ALS subjects comparing to analogue control tissues (P < 0.01), as well as in motor cortex of ALS subjects comparing to their sensory cortex (P < 0.05). In spinal cords the decrease in GST pi-mRNA expression was accompanied by a decrease of GST pi protein level. Results indicated lowered GST pi expression on both mRNA and protein levels in the regions of nervous system affected by ALS. The non-properly inactivated by GST toxic electrophiles and organic peroxides may thus contribute to motor neurons damage.