Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors

Summary The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the -methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly₇₄ of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.     

Acute post-irradiation canine intestinal blood flow

Radiation-induced early transient incapacitation (ETI) is accompanied by severe systemic hypotension, during which arterial blood pressure often decreases to less than 50 per cent of normal. One haemodynamic compensatory mechanism is increased peripheral resistance due to vasoconstriction. This vasoconstriction in the small intestine of dogs is disproportionately increased during haemorrhagic or endotoxic shock, and intestinal ischaemia is frequent. In an attempt to elucidate mechanisms underlying radiation-induced ETI and the gastrointestinal radiation syndrome, canine intestinal submucosal blood flow was measured by the hydrogen polarographic technique, both before and after exposure to gamma radiation. Systemic blood pressures, blood gases and haematocrits were determined simultaneously. Data obtained from 12 sham-irradiated dogs and 12 irradiated dogs indicated that 90 Gy, whole-body, gamma radiation produced a 31 per cent decrease in systemic mean blood pressure beginning within 20 min post-irradiation and lasting for at least 90 min. However, the intestinal submucosal blood flow did not decrease as anticipated, but it exhibited an actual post-irradiation increase. This increase in post-irradiation intestinal submucosal blood flow began within 5 min after irradiation and lasted for at least 90 min. Post-irradiation haematocrits were 10.5 per cent higher than those obtained before irradiation and those obtained from sham-irradiated subjects. Histopathological examination of ileal mucosa revealed significant pathologic lesions in some irradiated animals within two hours after exposure.     

Cultivation of aerobic chemoorganotrophic proteobacteria and gram-positive bacteria from a hot spring microbial mat.

The diversity of aerobic chemoorganotrophic bacteria inhabiting the Octopus Spring cyanobacterial mat community (Yellowstone National Park) was examined by using serial-dilution enrichment culture and a variety of enrichment conditions to cultivate the numerically significant microbial populations. The most abundant bacterial populations cultivated from dilutions to extinction were obtained from enrichment flasks which contained 9.0 x 10(2) primary producer (Synechococcus spp.) cells in the inoculum. Two isolates exhibited 16S rRNA nucleotide sequences typical of beta-proteobacteria. One of these isolates contained a 16S rRNA sequence identical to a sequence type previously observed in the mat by molecular retrieval techniques. Both are distantly related to a new sequence directly retrieved from the mat and contributed by a beta-proteobacterial community member. Phenotypically diverse gram-positive isolates genetically similar to Bacillus flavothermus were obtained from a variety of dilutions and enrichment types. These isolates exhibited identical 16S rRNA nucleotide sequences through a variable region of the molecule. Of the three unique sequences observed, only one had been previously retrieved from the mat, illustrating both the inability of the cultivation methods to describe the composition of a microbial community and the limitations of the ability of molecular retrieval techniques to describe populations which may be less abundant in microbial communities.     

Photosynthate Partitioning and Fermentation in Hot Spring Microbial Mat Communities

Patterns of (sup14)CO(inf2) incorporation into molecular components of the thermophilic cyanobacterial mat communities inhabiting hot springs located in Yellowstone National Park and Synechococcus sp. strain C1 were investigated. Exponentially growing Synechococcus sp. strain C1 partitioned the majority of incorporated (sup14)CO(inf2) into protein, low-molecular-weight metabolites, and lipid fractions (45, 22, and 18% of total incorporated carbon, respectively). In contrast, mat cores from various hot springs predominantly accumulated polyglucose during periods of illumination (between 77 and 85% of total incorporated (sup14)CO(inf2)). Although photosynthetically active, mat photoautotrophs do not appear to be rapidly growing, since we also detected only limited synthesis of macromolecules associated with growth (i.e., protein and rRNA). To test the hypothesis that polysaccharide reserves are fermented in situ under the dark anaerobic conditions cyanobacterial mats experience at night, mat cores were prelabeled with (sup14)CO(inf2) under illuminated conditions and then transferred to dark anaerobic conditions. Radiolabel in the polysaccharide fraction decreased by 74.7% after 12 h, of which 58.5% was recovered as radiolabeled acetate, CO(inf2), and propionate. These results indicate tightly coupled carbon fixation and fermentative processes and the potential for significant transfer of carbon from primary producers to heterotrophic members of these cyanobacterial mat communities.     

Two recurrent nonsense mutations and a 4 bp deletion in a quasi-symmetric element in exon 37 of the NF1 gene

We screened a total of 92 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exon 37 of the NF1 gene, by using temperature gradient gel electrophoresis. Two novel mutations were found: a 4 bp deletion in a so-called quasi-symmetric element (6789delTTAC) and a recurrent nonsense mutation, which was identified in two unrelated patients, at codon 2264 (C6792A). The independent origin of the latter mutation in two families was confirmed by haplotype analysis. The nonsense mutation and the 4 bp deletion are both predicted to lead to a truncated protein product lacking the Cterminal 20% (aproximately) of its sequence. The occurrence of three independent mutations among 92 NF1 patients at codons 2263–2264 (exon 37) suggests that a specific search for mutations in this area should be undertaken in screening programs for NF1 mutations.     

Chronopharmacokinetics and Cardiovascular Effects of Nifedipine

Orcadian phase dependency in pharmacokinetics and hemodynamic effects on blood pressure and heart rate of different galenic formulations of nifedipine (immediate-release, sustained-release, and i.v. solution) were studied in healthy subjects or in hypertensive patients. Pharmacokinetics of immediate-release but not sustained-release and i.v. nifedipine were dependent on time of day: immediate-release nifedipine had higher C_max (peak concentration) and shorter t_max (time-to-peak concentration) after morning than evening application, and bioavailibility in the evening was reduced by about 40%. Orcadian rhythm in estimated hepatic blood flow as determined by indocyanine green kinetics may contribute to these chronokinetics. A circadian time dependency was also found in nifedipine-induced effects on blood pressure and heart rate as monitored by 24-h ambulatory blood pressure measurements. In conclusion, the dose response relationship of oral nifedipine is influenced by the circadian organization of the cardiovascular system as well as by the galenic drug formulation.     

A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization

Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.     

Reflux induces DNA strand breaks and expression changes of MMP1+9+14 in a human miniorgan culture model

Gastroesophageal reflux disease has been implicated in the pathogenesis of adenocarcinoma of the oesophagus. The same applies to laryngopharyngeal reflux (LPR) and squamous cell cancer of the head and neck, but so far, this link has not been proven. The impact of low pH and bile acids has not been studied extensively in cells other than oesophageal cancer cell lines and tissue. The aims of this study were to investigate the pathogenic potential of reflux and its single components on the mucosa of the upper respiratory tract. We measured DNA stability in human miniorgan cultures (MOCs) and primary epithelial cell cultures (EpCs) in response to reflux by the alkaline comet assay. As matrix metalloproteinases (MMPs) are involved in extracellular matrix remodelling processes and may contribute to cancer progression, we studied the expression of MMP1, -9, and -14 in MOCs, EpC, UM-SCC-22B, and FADU_DD. DNA strand breaks (DNA-SBs) increased significantly at low pH and after incubation with human or artificial gastric juice. Single incubation with glycochenodeoxycholic acid also showed a significant increase in DNA-SBs. In epithelial cell cultures, human gastric juice increased the number of DNA-SBs at pH 4.5 and 5.5. Artificial gastric juice significantly up regulated the gene expression of MMP9. Western blot analysis confirmed the results of gene expression analysis, but the up regulation of MMP1, -9, and -14 was donor-specific. Reflux has the ability to promote genomic instability and may contribute to micro environmental changes suitable for the initiation of malignancy. Further functional gene analysis may elucidate the role of laryngopharyngeal reflux in the development of head neck squamous cell carcinoma (HNSCC).     

Exploring Sources of Emotional Distress among People Living with Scleroderma: A Focus Group Study

Background

Systemic sclerosis, or scleroderma, is a chronic and rare connective tissue disease with negative physical and psychological implications. Sources of emotional distress and the impact they have on the lives of people with scleroderma are not well understood.

Objectives

To gain an in-depth understanding of the emotional experiences and sources of emotional distress for women and men living with scleroderma through focus group discussions.

Methods

Three semi-structured focus group discussions were conducted (two in English, one in French) with a total of 22 people with scleroderma recruited through the Scleroderma Society of Ontario in Hamilton, Ontario and a scleroderma clinic in Montreal, Canada. Interviews were recorded, transcribed, and then coded for emerging themes using thematic inductive analysis.

Results

Core themes representing sources of emotional distress were identified, including: (a) facing a new reality; (b) the daily struggle of living with scleroderma; (c) handling work, employment and general financial burden; (d) changing family roles; (e) social interactions; and (f) navigating the health care system. Collectively, these themes refer to the stressful journey of living with scleroderma including the obstacles faced and the emotional experiences beginning prior to receiving a diagnosis and continuing throughout the participants’ lives.

Conclusion

Scleroderma was portrayed as being an unpredictable and overwhelming disease, resulting in many individuals experiencing multiple sources of emotional distress. Interventions and supportive resources need to be developed to help individuals with scleroderma and people close to them manage and cope with the emotional aspects of the disease.