Search

The metF gene in Escherichia coli and Salmonella typhimurium is under negative transcriptional control by the MetJ repressor. Expression of an S. typhimurium metF-lacZ gene fusion is repressed up to 10-fold by methionine addition to the growth medium in E. coli hosts encoding wild-type MetJ repressor; this repression is not seen in metJ mutants. metR mutations which eliminate the MetR activator protein result in two- to threefold-more-severe repression by the MetJ repressor. In a metJ metR double mutant, however, the level of metF-lacZ expression is the same as in a metJ mutant, suggesting that MetR antagonizes MetJ-mediated methionine repression of the metF promoter. A DNA footprint analysis showed that MetR binds to a DNA fragment carrying the metF promoter and protects two separate regions from DNase I digestion: a 46-bp region from position -50 to -95 upstream of the transcription initiation site and a 24-bp region from about position +62 to +85 downstream of the transcription initiation site and within the metF structural gene. Nucleotide changes in each of the MetR-binding sites away from the consensus sequence disrupt MetR-mediated regulation of the metF-lacZ fusion.

This pilot study evaluated the effectiveness and safety of a topically applied 2.5% solution of flutamide (a nonsteroidal antiandrogen) versus its placebo vehicle in the treatment of acne vulgaris. A total of 80 patients (33 males and 47 females) were treated for a period of 16 weeks. Inflammatory lesions (papules, pustules and nodules combined) responded better to flutamide (-56.7%) than to placebo (-36.7%; P=0.01). There was no meaningful difference between the effect of flutamide (-48.6%) and of placebo (-45.3%) on noninflammatory lesions (comedones). A relatively high level of local side-effects such as scaling, burning/stinging, pruritus, erythema and vesicles were observed in both the flutamide (60.0%) and the placebo (56.4%) groups. However, there were no systemic side-effects reported. This first clinical study demonstrated that flutamide 2.5% solution is effective in the topical treatment of acne vulgaris.

The vitamin B12 (B12)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product. It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism. Using Escherichia coli strains lysogenized with a lambda phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion. Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression. In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein. When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect. In a metJ mutant, B12 represses metE-lacZ expression less than twofold. When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored. The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-B12 holoenzyme.

Transcription of the metH gene in Salmonella typhimurium and Escherichia coli is positively regulated by the metR gene product, a DNA binding protein. The interaction between the MetR activator protein and the S. typhimurium metH control region was investigated. In vitro gel mobility shift assays and DNase I protection assays established that the MetR protein binds to and protects a 24-bp sequence in the metH promoter region from DNase I attack. This region includes the proposed metR recognition sequence 5'-TGAANNNNNCTCA-3'. Single-base-pair changes were introduced into the proposed MetR recognition sequence within the promoter region of a metH-lacZ gene fusion by oligonucleotide-directed mutagenesis. Two classes of mutations were identified. In the first class, the mutations caused reduced activation of the metH-lacZ fusions that correlated with reduced MetR binding. In the second class, activation of the metH-lacZ fusion was reduced, yet there was no appreciable reduction in MetR binding, indicating that the presence of bound MetR is not sufficient for activation of metH-lacZ gene expression. These two classes of mutations in the DNA binding site are grouped spatially, suggesting that the proposed MetR recognition sequence can be divided into two functional domains, one for binding and the other for activation.

We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that the mutant carries normal metE, metH, and metF genes; the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmid-borne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.

We used an in vitro DNA-dependent protein-synthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNA-dependent peptide-synthesizing system, synthesis of the di- and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site.

The DNA sequence of the Salmonella typhimurium metA control region is presented. S1 nuclease mapping was used to determine the transcription initiation site. By measuring beta-galactosidase levels in Escherichia coli strains lysogenized with lambda phage carrying a metA-lacZ gene fusion, the MetR protein was shown to activate the metA gene. Homocysteine, an intermediate in methionine biosynthesis, plays a negative role in the MetR-mediated activation mechanism. Gel mobility shift assays and DNase I protection experiments showed that the MetR protein binds to a DNA fragment carrying the metA control region and protects a 26-bp region beginning 9 bp upstream of the -35 promoter sequence.

Using an Escherichia coli lac deletion strain lysogenized with a lambda phage carrying a metH-lacZ gene fusion, we isolated trans-acting mutations that result in simultaneous 4- to 6-fold-elevated metH-lacZ expression, 5- to 22-fold-lowered metE-lacZ expression, and 9- to 20-fold-elevated metR-lacZ expression. The altered regulation of these genes occurs in the presence of high intracellular levels of homocysteine, a methionine pathway intermediate which normally inhibits metH and metR expression and stimulates metE expression. P1 transductions and complementation tests indicate that the mutations are in the metR gene. Our data suggest that the mutations result in an altered MetR activator protein that has lost the ability to use homocysteine as a modulator of gene expression.

The metR-mediated activation of the Salmonella typhimurium metE and metH genes was shown to be modulated by homocysteine, an intermediate in the methionine biosynthetic pathway. Homocysteine stimulates expression of a metE-lacZ gene fusion four- to fivefold by increasing transcription from the metE promoter. In contrast, homocysteine plays an inhibitory role in the metR-mediated activation of the metH gene, decreasing expression of a metH-lacZ gene fusion threefold.

This article discusses spirituality as an integral component of the occupational performance model. Spirituality is defined as the experience of meaning in everyday life activities. Some of the difficulties commonly encountered in operationalizing the concept of spirituality are discussed. Strategies built on minimizing or eliminating some of the difficulties are reviewed. The recommendations for practising therapists encompass suggestions for assessment, goal setting, and practice.

Incubation of human serum with either D- (1-14c)galactose (5 mM), D-(l-14c)glucose (5 mM) or L-(l-14c)fucose (5 mM) in vitro for 7 days under physiological conditions resulted in the accumulation of radioactivity into trichloroacetic acid precipitable material. Separation of the serum proteins by Sephadex G-200 chromatography, revealed the association of radioactivity with the. albumin fraction (95%) and to a lesser extent with IgG (4%) and IgM (1%). D-galactose glycosylated purified human IgG at 2 to 3 fold the rate of D-glucose or L-fucose. The rate of glycose incorporation into IgG increased parabolically with increasing pH and temperature of incubation, and followed a first order dependence with either the glycose or the IgG concentration. The post-translational modification of IgG through nonenzymatic glycosylation may affect its immunological properties in clinical conditions associated with increased blood sugar concentrations.

The DNA sequence of the Salmonella typhimurium glyA gene has been determined. The polypeptide deduced from the DNA sequence contains 417 amino acids and has a calculated molecular weight of 45428 daltons. S1 nuclease mapping experiments located the transcription start point and possible transcription termination region. The nucleotide and amino acid sequences for the S. typhimurium and Escherichia coli glyA genes were compared. The nucleotide sequences show 89% identity, and the amino acid sequences show 93% identity. In S. typhimurium there is an absence of REP sequences between the translation termination site and the proposed transcription termination site that are present in the E. coli sequence. A conserved sequence is found in both organisms extending from 79 to 117 bp upstream of the consensus -35 sequences of the glyA promoters. This conserved sequence shows homology to a sequence preceding the S. typhimurium metE gene determined to bind the MetR regulatory protein.

We isolated and characterized cis-acting mutations that affect the regulation of the metB gene of Salmonella typhimurium LT2. The mutations were isolated in an Escherichia coli lac deletion strain lysogenized with lambda bacteriophage carrying a metB-lacZ gene fusion (lambda JBlac) in which beta-galactosidase production is dependent upon metB gene expression. The mutant lysogens show elevated, poorly regulated beta-galactosidase production. The altered regulation is a result of disruption of the methionine control system mediated by the metJ repressor. The mutations are located in a region of dyad symmetry centered near the -35 sequence of the metB promoter. We propose that these mutations alter the repressor binding site and define the metB operator sequence. In addition, we discuss a highly conserved, nonsymmetric DNA sequence of unknown function which occurs in the control regions of the metA, metC, metE, metF, metG, and metJB genes of both S. typhimurium and E. coli.

The Salmonella typhimurium metE and metR genes share a common control region, with overlapping, divergently transcribed promoters. A double gene fusion was constructed in which the metE promoter directs expression of the Escherichia coli lacZ gene and the metR promoter directs expression of the E. coli galK gene. By using an E. coli strain lysogenized with a lambda bacteriophage carrying the metE-lacZ metR-galK double fusion (lambda Elac.Rgal), two classes of cis-acting mutations were isolated that increase metR-galK expression. The first class of mutations causes a simultaneous decrease in metE-lacZ expression by disrupting the normal MetR-mediated activation of the metE promoter. The mutations are located within a region extending from 17 to 34 base pairs upstream of the -35 region of the metE promoter. Gel mobility shift assays and DNaseI protection experiments demonstrated that the MetR protein specifically binds to a 24-base-pair region encompassing these mutations. The second class of mutations increases metR-galK expression by directly altering the promoter consensus sequences of the metE and metR promoters.

Regulation of the Salmonella typhimurium metR gene was studied by measuring beta-galactosidase levels in Escherichia coli strains lysogenic for a lambda bacteriophage carrying a metR-lacZ fusion. The results indicate that the metR gene is negatively regulated by its own gene product and that this autoregulation involves homocysteine as a corepressor. In addition, the results indicate that the metR gene is negatively regulated by the metJ gene product over a 70- to 80-fold range.

Regulation of the Salmonella typhimurium metJ gene was examined by measuring beta-galactosidase activity in Escherichia coli strains lysogenic for a phage carrying a metJ-lacZ gene fusion. The results indicated that the metJ gene is regulated by its own gene product and by methionine supplementation to the growth medium. This autoregulatory mechanism involved two tandem promoters, pJ1 and pJ2, separated by approximately 65 base pairs. Deletion analysis permitted the assessment of the activity of promoters pJ1 and pJ2 individually. Promoter Pj1 was negatively regulated by the metJ gene product and by methionine. Although Pj2 regulation remained unclear, evidence is presented which suggests that it is not negatively regulated like pJ1.

The article talks about the baby boomer population in Canada, focusing on workforce productivity, and disabilities found in older population. It states that programs like human resource management and organizational development can help in bringing social change. Occupational therapists should indulge in programs like occupational adaptation and occupational ecosystems for effectivity at workplaces. A flowchart is presented explaining the relationship between meaning and value contribution.

Efforts to develop more effective and shorter-course therapies for tuberculosis have included a focus on host-directed therapy (HDT). The goal of HDT is to modulate the host response to infection, thereby improving immune defenses to reduce the duration of antibacterial therapy and/or the amount of lung damage. As a mediator of innate and adaptive immune responses involved in eliminating intracellular pathogens, autophagy is a potential target for HDT in tuberculosis. Because Mycobacterium tuberculosismodulates mammalian target of rapamycin (mTOR) signaling to impede autophagy, pharmacologic mTOR inhibition could provide effective HDT. mTOR exists within two distinct multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2). Rapamycin and its analogs only partially inhibit mTORC1. We hypothesized that novel mTOR kinase inhibitors blocking both complexes would have expanded therapeutic potential. We compared the effects of two mTOR inhibitors: rapamycin and the orally available mTOR kinase domain inhibitor CC214-2, which blocks both mTORC1 and mTORC2, as adjunctive therapies against murine TB, when added to the first-line regimen (RHZE) or the novel bedaquiline-pretomanid-linezolid (BPaL) regimen. Neither mTOR inhibitor affected lung CFU counts after 4-8 weeks of treatment when combined with BPaL or RHZE. However, addition of CC214-2 to BPaL and RHZE was associated with significantly fewer relapses in C3HeB/FeJ compared to addition of rapamycin and, in RHZE-treated mice, resulted in fewer relapses compared to RHZE alone. Therefore, CC214-2 and related mTOR kinase inhibitors may be more effective candidates for HDT than rapamycin analogs and may have the potential to shorten the duration of TB treatment.

In this article, the author who is an occupational therapist talks about his interest in public policy. Topics discussed include his first work in public policy as the executive director of a social services agency in rural Alberta two years after completing graduation with a diploma in occupational therapy, development of cultural awareness programs for health-care institutions by him as part of his master's degree thesis work, and his belief in the value of community service.

Studies by Urbanowski et al. [Urbanowski, M. L., Stauffer, L. T., Plamann, L. S. & Stauffer, G. V. (1987) J. Bacteriol. 169, 1391-1397] have identified a regulatory locus, called metR, required for the expression of the metE and metH genes. We recently purified the MetR protein from Escherichia coli and showed that it could stimulate the in vitro expression of the metE gene and autoregulate its own synthesis. In the present study, the purified MetR protein has been shown to stimulate the in vitro expression of the metH gene. Also, the in vitro synthesized MetE, MetH, and MetR proteins were shown to be biologically active. The transcription start sites for the metE and metR genes have been determined, and DNA footprinting experiments have identified regions in the metE-metR intergenic sequence that are protected by either the MetR or MetJ proteins.

The nucleotide sequence of the Escherichia coli glyA gene has been determined. The amino acid sequence predicted from the DNA sequence consists of 417 residues. After the coding region there is a 185 nucleotide sequence preceding the proposed transcription termination region for the glyA gene. This region is preceded by a G‐C rich sequence that could form a stable stem–loop structure once transcribed, followed by an A–T rich sequence within which transcription appears to terminate. There is a long region of dyad symmetry and numerous smaller symmetrical regions between the site of translation termination and the proposed transcription termination region. These stem–loop structures show remarkable homology with intercistronic elements of other prokaryotic operons and may play a role in the regulation of glyA gene expression.

The nucleotide sequence of the Salmonella tqphirnurium metJ gene is presented along with the sequence of the promoter region for the closely linked metB gene. The two genes are transcribed in opposite directions, with transcription initiating from a single promoter for metB, and from two apparent promoters for metJ. RNA polymerase binding sites for metJ and metB, determined by in vitro protection studies, lie adjacent to each other and may overlap. The two metJ promoters, PJ1 and PJ2, are separated by approximately 65 base pairs. Binding of RNA polymerase in vitro could only be observed for PJ1, even though transcripts are initiated from both promoters in vivo. The metJ gene codes for a polypeptide of 105 amino acids with a calculated Mr of 12,110. The translation start site was determined by N-terminal amino acid sequence analysis of a metJ–slacZ fusion protein.

In Salmonella typhimurium the metE and metR promoters overlap and are divergently transcribed. Three tandem repeats of an 8 bp sequence defined previously as the metE operator site for MetJ-mediated repression also overlap the −35 region of the metR promoter. Starting with a metE-lacZ · metR-galK double gene fusion, site-directed mutagenesis was used to change nucleotides in each of the repeat units from the consensus sequence. Each mutation, along with the wild-type metE-lacZ · metR-galK gene fusion, was cloned into phage λgt2. Regulation of the metE and metR genes was examined by measuring β-galactosidase and galactokinase levels in Escherichia coli strains lysogenized with phage carrying the wild-type and mutant fusions. Mutations in each of the 8 bp repeat units disrupt MetJ-mediated repression for both the metE-lacZ and metR-galK gene fusions, suggesting that the metE and metR genes share a common operator site for the MetJ repressor.

There is a continuing debate within the health professions on the place of spirituality and the approach to be taken towards spiritual needs within holistic care. In occupational therapy, the need for both a working definition of spirituality and evidence to validate spirituality as a performance component has been identified (Urbanowski and Vargo 1994). The aim of this study was to add to the debate by exploring the experiences of occupational therapists who have encountered spiritual needs in their practice.A phenomenological approach using semi-structured interviews with three Christian occupational therapists explored their understanding of spirituality and experience of spiritual needs in practice. While the therapists interviewed had some difficulty in putting into words the concept of spirituality, they were able to identify spiritual needs in their clients and expressed opinions regarding the possible role of the occupational therapist in dealing with the spiritual needs of clients. It was clear that spiritual counselling was not considered to be appropriate for the occupational therapist, although recognising the spiritual dimension of an individual was important.In the United Kingdom, the occupational therapy profession needs to develop further its view on spirituality in holistic care and the impact that it has on the health and wellbeing of its clients.

Serine hydroxymethyltransferase from Escherichia coli was purified to homogeneity. The enzyme was a homodimer of identical subunits with a molecular weight of 95,000. The amino acid sequence of the amino and carboxy-terminal ends and the amino acid composition of cysteine-containing tryptic peptides were in agreement with the primary structure proposed for this enzyme from the structure of the glyA gene (M. Plamann, L. Stauffer, M. Urbanowski, and G. Stauffer, Nucleic Acids Res. 11:2065-2074, 1983). The enzyme contained no disulfide bonds but had one sulfhydryl group on the surface of the protein. Several sulfhydryl reagents reacted with this exposed group and inactivated the enzyme. Spectra of the enzyme in the presence of substrates and substrate analogs showed that the enzyme formed the same complexes and in similar relative concentrations as previously observed with the cytosolic and mitochondrial rabbit liver isoenzymes. Kinetic studies with substrates showed that the affinity and synergistic binding of the amino acid and folate substrates were similar to those obtained with the rabbit liver isoenzymes. The enzyme catalyzed the cleavage of threonine, allothreonine, and 3-phenylserine to glycine and the corresponding aldehyde in the absence of tetrahydrofolate. The enzyme was also inactivated by D-alanine caused by the transamination of the active site pyridoxal phosphate to pyridoxamine phosphate. This substrate specificity was also observed with the rabbit liver isoenzymes. We conclude that the reaction mechanism and the active site structure of E. coli serine hydroxymethyltransferase are very similar to the mechanism and structure of the rabbit liver isoenzymes.

The article looks at the U.S. National Park Service (NPS) and its expertise in conservation of protected areas. Particular focus is given to the Khor al-Adaid area in Qatar, also known as the Inland Sea, and the work of Fellows of the American Society of Landscape Architects (FASLA) Terri Urbanowski and Jodie Petersen on the program for its management and protection as of April 2014. Comments from David Krewson of the NPS's Office of International Affairs are included.

The article cites several studies related to bacteriology. One study found that expression of plasminogen activator in Escherichia coli has been achieved by chemically inducing stress responses. In another study scientists compared the overexpression of recombinant proteins in mutant strains of Escherichia coli BL21(DE3). In the third study researchers describe reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein. Researcher Mark L. Urbanowski and collaborators said that the Vibrio fischeri LuxR protein is the founding member of a family of acyl-homoserine lactone-responsive quorumsensing transcription factors.

This section presents news briefs related to the horticultural industry as of September 2010 including Green Lead Plants' release of its perennial catalog for 2010/2011 and Dow Agroscience's appointment of Mark Urbanowski as Portfolio Marketing Leader and Patrick Bell as Turf and Ornamental Product Manager.

Cells of the pathogenic bacterium Proteus mirabilisclump together to form colonies that, on meeting other colonies, can distinguish self from foreigners. Proteusdetects, then deters, interlopers of the same species using polypeptide weapons called proticines. Gibbs et al.investigated the genetic basis of this self-non-self recognition system in Proteusand discovered a locus of six genes that seem to constitute general and specific recognition determinants, as well as accessory genes. Within infected hosts, P. mirabilisinfections are usually clonal, so this recognition system could be a way for an established clone to prevent superinfection by another clone and to avoid competition. K. A. Gibbs, M. L. Urbanowski, E. P. Greenberg, Genetic determinants of self identity and social recognition in bacteria. Science321, 256-259 (2008). [Abstract][Full Text]

Profiles Alexandra Urbanowski, managing director of San Jose Repertory Theatre in San Jose, California. Job description; Career highlights and history; Personal background; Educational attainment.