The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm-free, CheY- containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo-taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.     

Influence of Lime and Organic Matter on the Mobility of Cadmium in Cadmium-Contaminated Soil in Relation to Nutrition of Spinach

Cadmium and cadmium compounds are water soluble, mobile in most soils, bio-available, and tend to bio-accumulate. A pot culture experiment was conducted on contaminated soil to study the influence of lime and organic matter on the mobility of cadmium in spinach and its rhizosphere soil. Application of lime (50% and 100% lime requirement) and organic matter (0.5 and 1% by weight of soil) to soil decreased the availability of Cd to the soil and plant throughout the crop growth. The highest diethylene triamine penta-acetic acid (DTPA) extractable Cd was 10.84 mg kg^?1 in the treatment OM₀ L₀ (No application of organic matter and lime) at 20 days after sowing of spinach. Likewise, the highest Cd concentration in spinach roots and shoots were 19.80 and 17.0 mg kg^?1 in the treatment OM₀ L₀ at 20 days after sowing. The Cd concentration in spinach roots and shoots were decreased by 63.23 and 71.88%, respectively, in the treatment OM₁ L₁₀₀ (application of FYM at 1.0% by weight of soil and lime at 100% lime requirement) after 60 days of growth. The lowest concentrations of Cd in the soil and plant after the harvest of the crop were 2.88 and 4.27 mg kg^?1, respectively, in the treatment OM₁ L₁₀₀ and resulted in 65.75 and 71.55% decrease over control (OM₀ L₀). The highest total chlorophyll content of leaves was 2.19 mg kg^?1 of fresh weight in the treatment OM₁ L₁₀₀ at 40 days of crop growth.     

Transactional Sex Risk across a Typology of Rural and Urban Female Sex Workers in Indonesia: A Mixed Methods Study

Context-specific typologies of female sex workers (FSWs) are essential for the design of HIV intervention programming. This study develops a novel FSW typology for the analysis of transactional sex risk in rural and urban settings in Indonesia. Mixed methods include a survey of rural and urban FSWs (n = 310), in-depth interviews (n = 11), key informant interviews (n = 5) and ethnographic assessments. Thematic analysis categorises FSWs into 5 distinct groups based on geographical location of their sex work settings, place of solicitation, and whether sex work is their primary occupation. Multiple regression analysis shows that the likelihood of consistent condom use was higher among urban venue-based FSWs for whom sex work is not the only source of income than for any of the other rural and urban FSW groups. This effect was explained by the significantly lower likelihood of consistent condom use by rural venue-based FSWs (adjusted OR: 0.34 95% CI 0.13–0.90, p = 0.029). The FSW typology and differences in organisational features and social dynamics are more closely related to the risk of unprotected transactional sex, than levels of condom awareness and availability. Interventions need context-specific strategies to reach the different FSWs identified by this study''s typology.     

Biochemical and Temporal Analysis of Events Associated with Apoptosis Induced by Lowering the Extracellular Potassium Concentration in Mouse Cerebellar Granule Neurons

Abstract: We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K⁺ concentration ([K⁺]_o), a significant part of the genomic DNA was already cleaved to high-molecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K⁺]_o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning ～4 h after the removal of high [K⁺]_o and developed to nuclear condensation 4 h later. Six hours after high [K⁺]_o deprivation, the DNA was fragmented into oligonucleosome-sized fragments. Within 6 h after removal of the extracellular K⁺, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K⁺]_o. Similar to high [K⁺]_o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K⁺]_o removal. An ～40% decrease in RNA and protein synthesis was detected by 6 h of [K⁺]_o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome-sized fragments, and the reduction in mitochondrial activity—events that occurred within the critical time—may indicate that these processes have an important part in the mechanism that committed the neurons to die.     

Studies toward a conjugate vaccine for anthrax. Synthesis and characterization of anthrose [4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-D-glucopyranose] and its methyl glycosides

The key step in the first chemical synthesis of anthrose (16) and its methyl alpha- (6) and beta-glycoside (22) was inversion of configuration at C-2 in triflates 10, 2, and 18, respectively, obtained from the common intermediate, methyl 4-azido-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside (1). To prepare methyl alpha-anthroside (6), methylation at O-2 of the gluco product 3, obtained from 2, was followed by hydrogenation/hydrogenolysis of the formed 2-methyl ether 4, to simultaneously remove the protecting benzyl group and reduce the azido function. Subsequent N-acylation of the formed amine 5 with 3-hydroxy-3-methylbutyric acid gave the target methyl alpha-glycoside 6. Synthesis of methyl beta-anthroside (22) comprised the same sequence of reactions, starting from the known methyl 4-azido-3-O-benzyl-4,6-dideoxy-beta-D-mannopyranoside (17), which was prepared from 1. In the synthesis of anthrose (16), 1-thio-beta-glucoside 11, obtained from 1 through 10, was methylated at O-2, and the azido function in the resulting benzylated 1-thioglycoside 12 was selectively reduced to give amine 13. After N-acylation with 3-hydroxy-3-methylbutyric acid, 1-thioglycoside 14 was hydrolyzed to give the corresponding reducing sugar, aldol 15, which was debenzylated to afford anthrose.     

Hsp70 translocates to the nuclei and nucleoli, binds to XRCC1 and PARP-1, and protects HeLa cells from single-strand DNA breaks

For many years, there has been uncertainty concerning the reason for Hsp70 translocation to the nucleus and nucleolus. Herein, we propose that Hsp70 translocates to the nucleus and nucleoli in order to participate in pathways related to the protection of the nucleoplasmic DNA or ribosomal DNA from single-strand breaks. The absence of Hsp70 in HeLa cells, via Hsp70 gene silencing (knockdown), indicated the essential role of Hsp70 in DNA integrity. Therefore, HeLa Hsp70 depleted cells were very sensitive in heat treatment and their DNA breaks were multiple compared to that of control HeLa cells. The molecular mechanism with which Hsp70 performs its role at the level of nucleus and nucleolus during stress was examined. Hsp70 co-localizes with PARP1 in the nucleus/nucleoli as was observed in confocal studies and binds to the BCRT domain of PARP1 as was revealed with protein–protein interaction assays. It was also found that Hsp70 binds simultaneously to XRCC1 and PARP-1, indicating that Hsp70 function takes place at the level of DNA repair and possibly at the base excision repair system. Making a hypothetical model, we have suggested that Hsp70 is the molecule that binds and interrelates with PARP1 creating the repair proteins simultaneously, such as XRCC1, at the single-strand DNA breaks. Our data partially clarify a previously unrecognized cellular response to heat stress. Finally, we can speculate that Hsp70 plays a role in the quality and integrity of DNA. Outlining prior scientific knowledge on the subject and novel information: The role of Hsp70 translocation to the nucleus and nucleolus during heat stress has been nearly unknown. It has been proposed that this biological phenomenon is correlated to Hsp70-chaperoning activity. Furthermore, some previous observations in yeast have revealed that Rad9 complexes—Rad9 being the prototype DNA-damage checkpoint gene—contain Ssa1 and or Ssa2 chaperone proteins, both reconstituting the functions of the corresponding Hsp70 in mammalian cells. Here, we propose that Hsp70 translocates to the nuclei/nucleoli during heat stress, binds to PARP-1 and/or XRCC1, and protects HeLa cells from increased single-strand DNA breaks.     

HS‐SPME‐GC‐FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2‐acetyl‐1‐pyrroline

A rapid micro‐scale solid‐phase micro‐extraction (SPME) procedure coupled with gas‐chromatography with flame ionized detector (GC‐FID) was used to extract parts per billion levels of a principle basmati aroma compound “2‐acetyl‐1‐pyrroline” (2‐AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre‐incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2‐AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 μg/kg. Along with 2‐AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)?2‐hexenal, pentadecanal, 4‐hydroxy‐2‐butanone, n‐hexanal, 2–6‐nonadienal, 3‐methoxy‐2(5H) furanone and 2‐acetyl‐1‐pyridine and octanal. High recovery of 2‐AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2‐AP production by B. cereus. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1356–1363, 2014     

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a ’shiro’. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.