Simple isolation of functional RNA from woody stems of gymnosperms

Stems of woody conifers contain high levels of polysaccharides and phenolic compounds that complicate the isolation of functional RNA from this highly lignified tissue. These difficulties were overcome by pulverizing the frozen tissue with a stainless-steel mortar and by effectively sequestering interfering phenolic compounds with vinylpyrrolidone polymers, thereby minimizing damage to nucleic acids during extraction. RNases were inhibited by aurintricarboxylic acid, and gelatinous polysaccharides were removed by inclusion of a 10% ethanol precipitation step. RNA was then isolated by precipitation with 33% isopropanol and ultracentrifugation onto a cushion of 5.7 M CsCl. Yields of 10 to 20 μg RNA/g FW were obtained from woody stems of several different gymnosperm species, including grand fir (Abies grandis), lodgepole pine (Pinus contorta), loblolly pine (Pinus taeda), Douglas fir (Pseudotsuga menziesii) and Pacific yew (Taxus brevifolia). The high quality of the RNA obtained was determined by UV-spectrophotometry, denaturing agarose gel electrophoresis, andin-vitro translation, and this material was used to construct cDNA libraries.     

A 96-well DNase I footprinting screen for drug–DNA interactions

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.     

Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.     

The structure and mechanism of methanol dehydrogenase

This is a review of recent work on methanol dehydrogenase (MDH), a pyrroloquinoline quinone (PQQ)-containing enzyme catalysing the oxidation of methanol to formaldehyde in methylotrophic bacteria. Although it is the most extensively studied of this class of dehydrogenases, it is only recently that there has been any consensus about its mechanism. This is partly due to recent structural studies on normal and mutant enzymes and partly due to more definitive work on the mechanism of related alcohol and glucose dehydrogenases. This work has also led to conclusions about the subsequent path of electrons and protons during the reoxidation of the reduced quinol form of the prosthetic group.     

Genetic background influences survival of infections with Salmonella enterica serovar Typhimurium in the Collaborative Cross

Salmonella infections typically cause self-limiting gastroenteritis, but in some individuals these bacteria can spread systemically and cause disseminated disease. Salmonella Typhimurium (STm), which causes severe systemic disease in most inbred mice, has been used as a model for disseminated disease. To screen for new infection phenotypes across a range of host genetics, we orally infected 32 Collaborative Cross (CC) mouse strains with STm and monitored their disease progression for seven days by telemetry. Our data revealed a broad range of phenotypes across CC strains in many parameters including survival, bacterial colonization, tissue damage, complete blood counts (CBC), and serum cytokines. Eighteen CC strains survived to day 7, while fourteen susceptible strains succumbed to infection before day 7. Several CC strains had sex differences in survival and colonization. Surviving strains had lower pre-infection baseline temperatures and were less active during their daily active period. Core body temperature disruptions were detected earlier after STm infection than activity disruptions, making temperature a better detector of illness. All CC strains had STm in spleen and liver, but susceptible strains were more highly colonized. Tissue damage was weakly negatively correlated to survival. We identified loci associated with survival on Chromosomes (Chr) 1, 2, 4, 7. Polymorphisms in Ncf2 and Slc11a1, known to reduce survival in mice after STm infections, are located in the Chr 1 interval, and the Chr 7 association overlaps with a previously identified QTL peak called Ses2. We identified two new genetic regions on Chr 2 and 4 associated with susceptibility to STm infection. Our data reveal the diversity of responses to STm infection across a range of host genetics and identified new candidate regions for survival of STm infection.     

Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis

Emerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal‐cysteine‐protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL‐mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non‐eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL‐mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non‐eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL‐stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL‐stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL‐primed osteoclastogenesis was observed in male and female CatB‐knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by P. gingivalis elevate RANKL‐primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.     

Protein degradation sets the fraction of active ribosomes at vanishing growth

Growing cells adopt common basic strategies to achieve optimal resource allocation under limited resource availability. Our current understanding of such “growth laws” neglects degradation, assuming that it occurs slowly compared to the cell cycle duration. Here we argue that this assumption cannot hold at slow growth, leading to important consequences. We propose a simple framework showing that at slow growth protein degradation is balanced by a fraction of “maintenance” ribosomes. Consequently, active ribosomes do not drop to zero at vanishing growth, but as growth rate diminishes, an increasing fraction of active ribosomes performs maintenance. Through a detailed analysis of compiled data, we show that the predictions of this model agree with data from E. coli and S. cerevisiae. Intriguingly, we also find that protein degradation increases at slow growth, which we interpret as a consequence of active waste management and/or recycling. Our results highlight protein turnover as an underrated factor for our understanding of growth laws across kingdoms.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

P2 pyridine N-oxide thrombin inhibitors: a novel peptidomimetic scaffold

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).