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T Parish

Publications and source records attributed to T Parish.

At least 19 recordsLinked to original sources
PubMed2004

The Mycobacterium tuberculosis dosRS two-component system is induced by multiple stresses.

Induction of the Mycobacterium tuberculosis dosR gene, which is known to respond to hypoxia, was measured using RTq-PCR following exposure to different stresses. Increased expression was seen after exposure to S-nitrosoglutathione (GSNO), ethanol and (to a lesser extent) H2O2, but not heat- or cold-shock. We also demonstrated that hspX, which is dependent on dosR for expression, is induced when cultures are left standing for 30 min, while significant but minor induction was seen following a 10 min centrifugation. Microarray analysis was used to compare gene expression in wild-type and deltadosR strains following 30 min standing. Fifty-two genes were significantly up-regulated, and 19 genes were down-regulated. These included genes that had previously been reported as being part of the dosR regulon, and also some novel ones.

DNA, Bacterial↗
PubMed2001

amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis.

BACKGROUND: The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation. RESULTS: We constructed a deletion mutant in one of the upstream ORFs (amiA). This mutant (Mad1) showed a constitutively high level of acetamidase expression. We identified four promoters in the upstream region using a beta-galactosidase reporter gene. One of these (P2) was inducible in the wild-type, but was constitutively active in Mad1. CONCLUSIONS: These results demonstrate that amiA encodes a negative regulatory protein which interacts with P2. Since amiA has homology to DNA-binding proteins, it is likely that it exerts the regulatory effect by binding to the promoter to prevent transcription.

Amidohydrolases↗
PubMed2001

Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality.

UNLABELLED: SWTTING: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome. OBJECTIVE: Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential. DESIGN: We took two strains of M. tuberculosis carrying the essential gene glnE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid. RESULTS: We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels. CONCLUSION: The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult or impossible using current methods.

Blotting, Southern↗
PubMed2001

Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates.

Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the metB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpD mutants were killed during this time. In SCID mice, both H37Rv and the metB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The proC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the metB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proC and trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the proC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.

Animals↗
PubMed2000

glnE is an essential gene in Mycobacterium tuberculosis.

Mycobacterium tuberculosis possesses a homologue of glnE, potentially encoding a regulator of glutamine synthetase activity. We attempted to construct glnE-disrupted mutants using a two-step strategy, whereby a single-crossover strain was first isolated, followed by sacB counterselection to isolate the double-crossover strain. Of 192 sucrose-resistant colonies tested, none were mutants, although the wild-type double crossover could be easily isolated. When a second copy of the wild-type glnE was integrated into the chromosome, we could isolate both wild-type and mutant double-crossover strains. Thus, the chromosomal gene could only be replaced with a disrupted copy when another functional copy of the gene was provided, demonstrating that this gene is essential under the conditions tested.

Crossing Over, Genetic↗
PubMed1999

Mycobacteria: bugs and bugbears (two steps forward and one step back).

The use of molecular techniques to study the mycobacteria has advanced greatly since the first genomic libraries of Mycobacterium tuberculosis and M. leprae were constructed in 1985. However, there are still pitfalls for the unwary. Most of the problems associated with the use of molecular techniques to study mycobacteria can be related to one of the following problems: slow growth rate causing problems with contamination; the formation of macroscopic clumps when grown in culture; resistance to standard chemical lysis procedures; the requirement for containment facilities for pathogenic species; the lack of suitable genetic vectors; and the problems of spontaneous antibiotic resistance. Despite these problems, considerable progress has been made and standard techniques have been developed for the preparation of protein, nucleic acids (DNA and RNA) and cell wall components, chemical and transposon mutagenesis and gene replacement methods, the use of reporter genes and expression vectors, and improved detection and drug sensitivity testing.

Bacteriological Techniques↗
PubMed1998

An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis.

A novel expression vector utilising the highly inducible acetamidase promoter of Mycobacterium smegmatis was constructed. High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide. The recombinant protein could be simply and efficiently purified from the bacterial sonicate by virtue of a C-terminal 6-histidine tag, demonstrating that this purification strategy can be used for the mycobacteria. The histidine tag had no apparent effect on the protein conformation or immunogenicity, suggesting that the vector described may prove useful for the purification of native-like recombinant mycobacterial proteins from fast-growing mycobacterial hosts.

Amidohydrolases↗
PubMed1997

Development and use of a conditional antisense mutagenesis system in mycobacteria.

Expression from a 2.3 kb region upstream of the inducible acetamidase gene from Mycobacterium smegmatis was shown to be upregulated by acetamide. A DNA fragment containing the start of the M. smegmatis hisD gene was cloned in front of the promoter, such that the antisense message was produced. When this construct was induced in vivo, the bacteria became phenotypically histidine auxotrophs; this auxotrophy was restored by histidine supplementation. Auxotrophy was not observed under non-induced conditions. Antisense mutagenesis may be useful for observing the phenotypic inactivation of specific mycobacterial genes, and an inducible system such as that described would allow the study of essential genes.

Acetamides↗
PubMed1997

Palatal rugae patterns in Australian aborigines and Caucasians.

The purpose of this study was to determine whether rugae patterns change with age and to compare the number and pattern of rugae in Australian Aborigines with those of Caucasians. For the longitudinal part of the study, serial dental casts of ten Aborigines, from 6 to 20 years of age, were examined and rugae patterns were recorded. To enable comparisons to be made between different ethnic groups an additional 100 dental casts of Australian Aborigines and 200 casts of caucasians, ranging in age from 13 to 17 years, were examined. Characteristics observed were number, length, shape, direction and unification of rugae. The length of rugae increased significantly with age but the total number of rugae remained constant. Thirty-two per cent of rugae showed changes in shape, while 28 per cent displayed a change in orientation. In contrast to studies suggesting that rugae move forward with age, the majority of Aboriginal rugae that changed direction moved posteriorly. Changes in rugae patterns have been assumed to result from palatal growth but alterations in pattern were observed in the Aboriginal sample even after palatal growth had ceased. The mean number of primary rugae in Aborigines was higher than in Caucasians, although more primary rugae in Caucasians exceeded 10 mm in length than in Aborigines. The most common shapes in both ethnic groups were wavy and curved forms, whereas straight and circular types were least common. There was a statistically significant association between rugae forms and ethnicity, straight forms being more common in Caucasians whereas wavy forms were more common Aborigines.

Adolescent↗
PubMed1997

A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability.

A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes.

Cell Membrane Permeability↗
PubMed1994

The application of luciferase as a reporter of environmental regulation of gene expression in mycobacteria.

This paper describes the construction of pSG10, the first mycobacterial promoter probe shuttle vector to use the structural gene of a bacterial luciferase as a reporter gene. To examine the utility of using bacterial luciferase to measure gene expression in mycobacteria, the authors have used this vector to monitor the induction of the acetamidase gene promoter of Mycobacterium smegmatis. Luciferase proved to be a rapid, sensitive and easily assayable reporter of changes in gene activity in response to environment in mycobacteria.

Acetamides↗
PubMed1994

Relative effects of alpha 1-adrenoceptor blockade, converting enzyme inhibitor therapy, and angiotensin II subtype 1 receptor blockade on ventricular remodeling in the dog.

BACKGROUND: Progressive ventricular remodeling after myocardial damage is associated with a poor prognosis. Optimal prevention of the histopathological processes involved in remodeling requires a more complete understanding of the mechanisms involved in initiating and maintaining these structural changes. Since the sympathetic nervous system and the renin-angiotensin system may be involved in the remodeling process, the structural effects of pharmacological inhibitors have been evaluated in a canine model of localized myocardial injury resulting from transmyocardial DC shock. METHODS AND RESULTS: The study is comprised of two protocols run in series. In protocol 1, zofenopril (Z), a converting enzyme inhibitor (CEI), prevented the increase in left ventricular mass (LVM) and end-diastolic volume (LVV) observed in the control group (C) at 16 weeks (Z: LVM, 69.8 +/- 3.4 to 65.4 +/- 2.6 g, P = NS; LVV, 45.4 +/- 2.7 to 51.6 +/- 2.7 mL, P = NS; C: LVM, 68.4 +/- 3.2 to 91.4 +/- 2.9 g, P = .0001; LVV, 56.6 +/- 3.0 to 71.9 +/- 2.4 mL, P = .0003). Terazosin, an alpha 1-adrenoceptor antagonist, failed to prevent remodeling at 16 weeks despite continued receptor blockade. In protocol 2, the antiremodeling effect of full-dose CEI therapy with ramipril was confirmed. Low-dose ramipril that exerted no hemodynamic effect failed to prevent remodeling (LVM, 89.7 +/- 4.6 to 105.7 +/- 3.4 g, P = .01; LVV, 61.8 +/- 3.8 to 76.8 +/- 3.3 mL, P = .002). An angiotensin II subtype 1 receptor blocker also failed to prevent the increase in LVM or LVV (LVM, 89.0 +/- 4.6 to 109.7 +/- 5.3 g, P = .0001; LVV, 66.0 +/- 1.9 to 78.4 +/- 3.6 mL, P = .007). CONCLUSIONS: High-dose CEI therapy can prevent progressive structural changes resulting from localized myocardial damage induced by DC shock. the failure of alpha 1-adrenoceptor blockade and angiotensin II subtype 1 blockade to attenuate remodeling argues against an important direct role for norepinephrine acting through alpha 1-receptors or angiotensin II acting through the type 1 receptor in the remodeling process in this model.

Adrenergic alpha-Antagonists↗
PubMed1987

Identification and androgen regulation of two proteins released by T47D human breast cancer cells.

Three [35S]methionine-labeled polypeptides released by T47D human breast cancer cells have been identified as corresponding to two proteins previously described in breast gross cystic disease fluid. A Mr 43,000 protein was immunoprecipitated by polyclonal antibodies to the Zn-alpha 2-glycoprotein. A Mr 18,000 and a Mr 13,000 polypeptide were both immunoprecipitated by four monoclonal antibodies directed against four separate epitopes of Mr 15,000 gross cystic disease fluid protein, a major protein that is characteristic of apocrine gland secretions. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified Mr 15,000 gross cystic disease fluid protein migrated at the level of the Mr 18,000 protein of T47D cells. The proteins were regulated by androgens and progestins. In addition to a general stimulation of protein secretion, 5 alpha-dihydrotestosterone (DHT) specifically increased, by 2- to 20-fold, the release of the Mr 43,000 and Mr 18,000 proteins into the medium. DHT also increased the cellular level of the Mr 18,000 protein, as shown by immunoprecipitation with a Mr 15,000 gross cystic disease fluid protein antibody, which suggests a stimulation of protein synthesis. The progestin 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione also increased the release in the medium of the Mr 43,000 and Mr 18,000 proteins, but higher molar concentrations were required than in the case of DHT. The induction of these proteins by DHT and 17,21-dimethyl-19nor,4,9-pregnadiene-3,20-dione was specifically inhibited by the antiandrogen flutamide which has no effect on other progestin-regulated proteins. This suggests an effect mediated by the androgen receptor. This is the first report on the identification of two proteins induced by androgens in a human breast cancer cell line. These proteins should be useful in studying the role of androgens in human mammary tumors and their mechanism of action in cell culture.

Androgens↗
PubMedDate not supplied

Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis.

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.

Colony Count, Microbial↗