The variable subdomain of Escherichia coli SecA functions to regulate SecA ATPase activity and ADP release

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi(r)) and suppression of signal sequence defects (PrlD). The SecAΔ(519-547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.     

Diversity and phylogenetic relationships of Wolbachia in Drosophila and other native Hawaiian insects

Wolbachia is a genus of parasitic alphaproteobacteria found in arthropods and nematodes, and represents on of the most common, widespread endosymbionts known. Wolbachia affects a variety of reproductive functions in its host (e.g., male killing, cytoplasmic incompatibility, parthenogenesis), which have the potential to dramatically impact host evolution and species formation. Here, we present the first broad-scale study to screen natural populations of native Hawaiian insects for Wolbachia, focusing on the endemic Diptera. Results indicate that Wolbachia infects native Hawaiian taxa, with alleles spanning phylogenetic supergroups, A and B. The overall frequency of Wolbachia incidene in Hawaiian insects was 14%. The incidence of infection in native Hawaiian Diptera was 11% for individuals and 12% for all species screened. Wolbachia was not detected in two large, widespread Hawaiian dipteran families—Dolichopodidae (44 spp screened) and Limoniidae (12 spp screened). Incidence of infection within endemic Hawaiian lineages that carry Wolbachia was 18% in Drosophilidae species, 25% in Caliphoridae species, > 90% in Nesophrosyne species, 20% in Drosophila dasycnemia and 100% in Nesophrosyne craterigena. Twenty unique alleles were recovered in this study, of which 18 are newly recorded. Screening of endemic populations of D. dasycnemia across Hawaii Island revealed 4 unique alleles. Phylogenetic relationships and allele diversity provide evidence for horizontal transfer of Wolbachia among Hawaiian arthropod lineages.     

Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway.

Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.     

Investigating the effect of increasing robot group sizes on the human psychophysiological state in the context of human–swarm interaction

We study the psychophysiological state of humans when exposed to robot groups of varying sizes. In our experiments, 24 participants are exposed sequentially to groups of robots made up of 1, 3 and 24 robots. We measure both objective physiological metrics (skin conductance level and heart rate), and subjective self-reported metrics (from a psychological questionnaire). These measures allow us to analyse the psychophysiological state (stress, anxiety, happiness) of our participants. Our results show that the number of robots to which a human is exposed has a significant impact on the psychophysiological state of the human and that higher numbers of robots provoke a stronger response.     

Loss of Acid Sensing Ion Channel-1a and Bicarbonate Administration Attenuate the Severity of Traumatic Brain Injury

Traumatic brain injury (TBI) is a common cause of morbidity and mortality in people of all ages. Following the acute mechanical insult, TBI evolves over the ensuing minutes and days. Understanding the secondary factors that contribute to TBI might suggest therapeutic strategies to reduce the long-term consequences of brain trauma. To assess secondary factors that contribute to TBI, we studied a lateral fluid percussion injury (FPI) model in mice. Following FPI, the brain cortex became acidic, consistent with data from humans following brain trauma. Administering HCO₃ ⁻ after FPI prevented the acidosis and reduced the extent of neurodegeneration. Because acidosis can activate acid sensing ion channels (ASICs), we also studied ASIC1a^−/− mice and found reduced neurodegeneration after FPI. Both HCO₃ ⁻ administration and loss of ASIC1a also reduced functional deficits caused by FPI. These results suggest that FPI induces cerebral acidosis that activates ASIC channels and contributes to secondary injury in TBI. They also suggest a therapeutic strategy to attenuate the adverse consequences of TBI.     

Patterns of DNA methylation in the normal colon vary by anatomical location,gender, and age

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.     

Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca²⁺ concentration required for PLI and PLII activity was determined to be 0·5 mmol l⁻¹. The Ca²⁺ requirement could not be replaced by other metal cations such as Mg²⁺, Cu²⁺, Zn²⁺, Fe³⁺ and Co²⁺. Optimal pH for activity was determined to be between 8·5 and 9·0. The K _m values for sodium polygalacturonate were 1·28 and 1·11 mg ml⁻¹ for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl₂ or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.     

Catheter-tract infections in rhesus macaques (Macaca mulatta) with indwelling intravenous catheters

Development of catheter-tract infections in experimental animals can have devastating consequences on animal health and the functional lifespan of surgical implants. To measure the incidence of catheter-tract infections in animals with exteriorized intravenous catheters in this facility and assess the effects of these infections on mean catheter lifespan, health records of 31 Macaca mulatta with catheters were reviewed. Records spanned the interval of January 1, 1996 through October 1, 1997. Catheter-tract infections in 16 of 53 (30.2%) monkeys with catheters were diagnosed based on a combination of clinical signs of infection and results of bacterial culture. Segmental catheter-tract infections reduced mean catheter lifespan to 147 days, compared with 354 days for uninfected catheters. Exit-wound, local tunnel, and surgical-site infections did not significantly reduce catheter lifespan. Bacterial culture reports documented 31 isolates; 41.9% (13 of 31) were coagulase-negative staphylococci, and 22.6% (7 of 31) were Staphylococcus aureus. Of 20 isolates tested, 15 (75%) were resistant to methicillin/oxacillin in vitro. Antimicrobial susceptibility testing of methicillin-resistant and methicillin-susceptible isolates indicated that, compared with methicillin-sensitive isolates, methicillin-resistant isolates had a pattern of multiple antibiotic resistance. Catheter-tract infections were common in this colony of rhesus macaques, and clinically severe infections caused a drastic reduction in catheter lifespan. Approximately half (48%) the bacterial isolates were methicillin-resistant gram-positive bacteria.     

Effects of growth rate and influent substrate concentration on effluent quality from chemostats containing bacteria in pure and mixed culture

Studies were performed using pure cultures of A. acrogenes and E. coli and a heterogeneous microbial population growing in carbon-limited chemostats with glucose as the sole carbon and energy source. A two-level factorial experimental design was employed to test the hypothesis that the concentration of growth-limiting substrate in a chemostat is controlled by the growth rate alone and is independent of the concentration of substrate entering the reactor. The pure culture experiments showed that the conclusions depend upon the measurement employed for growth-limiting substrate. When the concentration of glucose was measured directly, the hypothesis was found to be true within the limits of the study (500–1500 mg/liter). However, if the chemical oxygen demand (COD) test was used as the measure of growth-limiting substrate the hypothesis was found to be false. When heterogeneous cultures were employed the hypothesis was false regardless of the technique used to measure the concentration of growth-limiting substrate. Nevertheless, it was possible to generate regression equations which described the interactions among influent COD, growth rate, and effluent COD with a high level of correlation.     

Alterations in glycosaminoglycan metabolism in β-aminopropionitrile-treated chick embryos

1. Na(2) (35)SO(4), [1-(14)C]glucosamine and [1-(14)C]acetate were used as precursors of the sulphated glycosaminoglycans to study the biochemical effect of beta-aminopropionitrile in chick embryos. The incorporation of all three precursors was decreased in the treated embryos between days 7 and 10 of embryonic development. No inhibition of incorporation of these precursors occurred between days 16 and 20 of embryonic development at the dosages of beta-aminopropionitrile used. 2. beta-Aminopropionitrile treatment also decreased the amount of N-acetylhexosamines in the chick embryo and decreased the percentage of the hexosamine esterified by nucleotides. Respiration was decreased by homogenates prepared from treated embryos. Likewise, UDP-xylosyl- and UDP-galactosyl-transferase activities were decreased in treated embryos and cartilage from embryos and growing chicks. 3. The data suggest that beta-aminopropionitrile, in addition to the known lathyrogenic activity, either is or gives rise to a potent metabolic poison that interferes with basic cellular metabolism. The results are consistent with a decreased rate of ATP generation as an explanation for the decrease in glycosaminoglycan synthesis.