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P Dennis

Publications and source records attributed to P Dennis.

At least 19 recordsLinked to original sources
PubMed2003

Free RNA polymerase and modeling global transcription in Escherichia coli.

Growth rate-dependent changes in the cytoplasmic concentration of free functional RNA polymerase, [R(f)], affect the activity of all bacterial genes. Since [R(f)] is not accessible to direct experimental quantitation, it can only be found indirectly from an evaluation of promoter activity data. Here, a theory has been derived to calculate [R(f)] from the concentrations of total RNA polymerase and promoters in a model system with known Michaelis-Menten constants for the polymerase-promoter interactions. The theory takes transcript lengths and elongation rates into account and predicts how [R(f)] changes with varying gene dosages. From experimental data on total concentrations of RNA polymerase and kinetic properties of different classes of promoters, the theory was developed into a mathematical model that reproduces the global transcriptional control in Escherichia coli growing at different rates. The model allows an estimation of the concentrations of free and DNA-bound RNA polymerase, as well as the partitioning of RNA polymerase into mRNA and stable RNA synthesizing fractions. According to this model, [R(f)] is about 0.4 and 1.2 microM at growth rates corresponding to 1.0 and 2.5 doublings/h, respectively. The model accurately reflects a number of further experimental observations and suggests that the free RNA polymerase concentration increases with increasing growth rate.

DNA, Bacterial↗
PubMed2002

Kinetic properties of rrn promoters in Escherichia coli.

How do bacteria adapt and optimize their growth in response to different environments? The answer to this question is intimately related to the control of ribosome bio-synthesis. During the last decades numerous proposals have been made to explain this control but none has been definitive. To readdress the problem, we have used measurements of rRNA synthesis rates and rrn gene dosages in E. coli to find the absolute transcription rates of the average rrn operon (transcripts per min per operon) at different growth rates. By combining these rates with lacZ expression data from rRNA promoter-lacZ fusions, the abolute activities of the isolated rrnB P1 and P2 promoters were determined as functions of the growth rate in the presence and absence of Fis and of the effector ppGpp. The promoter activity data were analyzed to obtain the relative concentrations of free RNA polymerase, [R(f)], and the ratio of the Michaelis-Menten parameters, V(max)/K(m) (promoter strength), that characterize the promoter-RNA polymerase interaction. The results indicate that changes in the basal concentration of ppGpp can account for all growth-medium dependent regulation of the rrn P1 promoter strength. The P1 promoter strength was maximal when Fis was present and the level of ppGpp was undetectable during growth in rich media or in ppGpp-deficient strains; this maximal strength was 3-fold reduced when Fis was removed and the level of ppGpp remained undetectable. At ppGpp levels above 55 pmol per cell mass unit (OD(460)) during growth in poor media, the P1 promoter strength was minimal and not affected by the presence or absence of fis. The half-maximal value occurred at 20 pmol ppGpp/OD(460) and corresponds to an intracellular concentration of about 50 microM. In connection with previously published data, the results suggest that ppGpp reduces the P1 promoter strength directly, by binding RNA polymerase, and indirectly, by inhibiting the synthesis of Fis.

DNA, Ribosomal↗
PubMed2001

Cimetidine in painful bladder syndrome: a histopathological study.

OBJECTIVE: To determine whether oral cimetidine, which reportedly improves symptoms in 60-70% of patients with painful bladder syndrome/interstitial cystitis has a similar mechanism of action on the human bladder and involving a similar peptidergic pathway as it has in human stomach, where it alters histamine-gastrin reactions mediated via H2 receptors and a proton pump. PATIENTS AND METHODS: Fourteen patients (13 women and one man, mean age 51 years) with refractory bladder pain and irritative urinary symptoms were treated with cimetidine. The response to cimetidine was assessed by outpatient follow-up and an analogue pain score. Bladder biopsies from eight patients were stained with haematoxylin and eosin, and a polyclonal antibody to gastrin, with counterstaining using toluidine blue, to detect mast cell granules containing histamine. Biopsies from normal areas of the bladder from an age-matched control group of patients with transitional cell carcinoma of the bladder were stained similarly. Human stomach and colon were used as positive controls for gastrin and toluidine blue, respectively. The clinical response to cimetidine was compared with the histology in a blinded fashion. RESULTS: Eight of the patients responded well to cimetidine; none of the biopsies showed evidence of carcinoma in situ. Although G cells in the stomach stained well for gastrin none of the bladder biopsies showed gastrin-like immunoreactivity, apart from some nonspecific urothelial staining. Numerous mast cells with crimson granules and pale nuclei were visible in three patients and fewer in three others; their presence or absence did not correlate with the symptomatic response. CONCLUSIONS: Cimetidine is a useful medical treatment for bladder pain but the presence or absence of gastrin or histamine-like immunoreactivity does not explain its therapeutic benefit.

Cimetidine↗
PubMed2001

Cytoplasmic RNA Polymerase in Escherichia coli.

To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase beta and beta' subunits in DNA-free minicells from the minicell-producing Escherichia coli strain chi925 was determined. In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase. The concentration of cytoplasmic RNA polymerase beta and beta' subunits in minicells produced by this strain corresponded to about 17% (or 2.5 microM) of the value found in whole cells. Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell. On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive. RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay. Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.

Bacteriophage T7↗
PubMed2000

mRNA composition and control of bacterial gene expression.

The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of beta-galactosidase expressed from three different promoter-lacZ fusions (P(spc)-lacZ, P(RNAI)-lacZ, and P(RNAII)-lacZ) was measured (i) in a relA(+) background during exponential growth at different rates and (ii) in relA(+) and DeltarelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp.

Bacterial Proteins↗
PubMed1999

Activities of constitutive promoters in Escherichia coli.

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain. The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains.

Cell Division↗
PubMed1999

Decay of rplN and lacZ mRNA in Escherichia coli.

To examine the previously proposed retroregulation model of spc mRNA degradation, two strains of Escherichia coli B/r were used; one has wild-type spc and lac operons and the other has a lac operon deletion, a wild-type spc operon, and a Pspc-rplN-lacZ fusion operon lacking the normal control sites of the spc operon (rplN is the first gene in the spc operon of ribosomal proteins). The decay of rplN mRNA and of lacZ mRNA in these strains was determined during exponential growth at different rates and after transcript initiation was inhibited by the antibiotic rifampicin. Functional decay of lacZ mRNA was monitored by measurements of beta-galactosidase activity and chemical decay was monitored using probes complementary to rplN, rplX, and to the 5' and 3'-terminal sections of lacZ. Analysis of the data was based on the assumption that the decay involves an endonucleolytic cleavage that functionally inactivates the mRNA and that this is followed by exonucleolytic degradation of the cleavage products. The major conclusions were: (1) During exponential growth, lacZ mRNA of the lac operon was translated about twice as frequently as lacZ mRNA of the spc-lac fusion, and both kinds of lacZ mRNA were translated at an elevated rate in the presence of rifampicin. (2) For lacZ mRNA from the lac operon, the endonuclease inactivation reaction was not affected by rifampicin, but the exonuclease reaction was inhibited. (3) The decay of rplN mRNA from the spc operon was accelerated in the presence of rifampicin; the average life was estimated to be six minutes during exponential growth in LB medium, and 2.8 minutes in the presence of rifampicin. (4) The decay of the rplN section of mRNA from the spc-lac operon fusion was coupled to the decay of the downstream lacZ mRNA section and was strongly inhibited (i.e. partially blocked) in the presence of rifampicin. These results show that the decay of spc mRNA differs in some important aspects from the decay of lac mRNA and support the retroregulation model. Moreover, the results indicate that rifampicin can have a significant and selective impact on the kinetics of both mRNA translation and decay.

Escherichia coli↗
PubMed1997

Multiple nuclear proteins bind a novel cis-acting element that regulates the muscle-specific expression of the mouse nicotinic acetylcholine receptor alpha-subunit gene.

Expression of the nicotinic acetylcholine receptor (AChR) is transcriptionally regulated during the development of vertebrate striated muscle. To better define regulatory elements involved in this process, site-directed mutations were made in the gene's 86 bp muscle specific enhancer. Transient expression assays in skeletal muscle C2C12 cells indicated that all three E-boxes, plus a novel sequence outside the E-boxes, are necessary for full activity of the AChR gene in myotubes. Gel mobility shift assays demonstrated that mutations in the non-E-box sequence disrupted the formation of two DNA/protein complexes while not affecting myoD binding. Methylation interference footprinting confirmed that the complexes form at nucleotides within the mutated region, and also include part of the central E-box. UV crosslinking of nuclear proteins to a DNA probe identified five proteins of 125, 81, 55, 42, and 35 kDa that bind to this region; with the 125 kDa protein being differentially bound in U.V. crosslink assays during the transition from myoblasts to myotubes. These data suggest that interactions between this DNA element and the five proteins contribute to the transcriptional control of the AChR alpha-subunit gene expression during the differentiation of skeletal muscle.

Animals↗
PubMed1993

Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

Alcohol Dehydrogenase↗
PubMed1992

An inter-observer and intra-observer variability study on the diagnosis of lymph node biopsy specimens.

One hundred lymph node biopsy specimens were examined on two separate occasions by seven pathologists differing in experience in lymphoreticular pathology. Neither history nor immunohistochemistry was provided and the study, therefore, focused on morphological interpretation alone. The participants evaluated each case using a constructed response form in which the confidence with which they entered each response was also entered. Agreement on various points, between pathologists, between the two rounds, and with the referring centre was assessed. Whilst there was a high level of agreement over a diagnosis of benign vs. malignant and non-Hodgkin lymphoma vs. Hodgkin's disease, there was considerably less agreement over both T vs. B cell phenotype and high vs. low grade. The lack of agreement over grade, an evaluation which is usually made independent of immunohistochemistry, is particularly important, because of the relevance to selection of treatment. Proliferation markers may be more appropriate determinants of treatment choice.

Biopsy↗
PubMed1990

Growth factor control of extracellular proteolysis.

The involvement of proteases and growth factors in angiogenesis is complex. The angiogenic factor basic fibroblast growth factor (bFGF) induces increased synthesis of both plasminogen activator and collagenase in endothelial cells. In addition, bFGF increases the number of plasminogen activator receptors on the cell surface. Increased production of plasmin may be responsible for the release of soluble complexes of heparan sulfate-bFGF which may be the active form of bFGF. The activity of a negative regulator of angiogenesis, transforming growth factor beta (TGF-beta), is also regulated by proteases since the released latent form of TGF-beta is activated by a surface proteolytic assembly plasminogen activator and plasmin. Since TGF-beta induces an inhibitor of plasminogen activator, the activation reaction is self-regulatory.

Animals↗
PubMed1986

Archaebacteria: transcription and processing of ribosomal RNA sequences in Halobacterium cutirubrum.

The chromosome of Halobacterium cutirubrum contains a single ribosomal RNA gene cluster. The 5' to 3' organization of genes within this 6-kpb region is: 16S, alanine tRNA, 23S, 5S, cysteine tRNA. The entire gene cluster is transcribed as a single long primary transcript; processing of mature RNA sequences from the 5' region of the transcript begins prior to the completion of synthesis at the 3' end. There are five conserved octanucleotide direct repeats (TGCGAACG) in the 900-bp 5'-flanking sequence in front of the 16S gene. The positions of these repeat sequences correspond to the different 5' ends of the primary transcript and probably represent the RNA polymerase start sites. The 16S and 23S rRNA genes are surrounded by long nearly perfect inverted repeat sequences. These sequences probably form duplex structures in the primary transcript and are recognized by an RNaseIII-like endonuclease activity that carries out the initial excision of the precursor 16S and 23S rRNA sequences. These precursors are rapidly trimmed tot he mature 16S and 23S molecules and assembled into ribosomal particles. The processing sites for 5S rRNA appear to be at or very near to the mature ends of the 5S molecule. The tRNA sequences are processed with reduced efficiency from the primary transcript. Nuclease cuts have been detected at the ends as well as in the middle of the cysteine tRNA sequence suggesting that there may be alternative processing pathways, one resulting in proper excision of the mature tRNA sequence and the other resulting in improper excision and degradation of the tRNA sequence. The transcription termination sequence is believed to be at or beyond an AT-rich sequence preceded by a GC-rich sequence located distal to the cysteine tRNA gene.

Base Sequence↗
PubMed1986

Preservative cytotoxicity to cultured corneal epithelial cells.

Cultured human and rat corneal epithelial cells with 51Cr incorporated were used as a model to test the cytolytic action of four common preservatives. Benzalkonium chloride, chlorohexidine and thimerosol were all found to lyse greater than 40% cells when incubated for fifteen minutes at concentrations in clinical use in topical ophthalmic medications. Chlorobutanol is the only preservative tested which has a low level of cytotoxicity (10%) and which, under these conditions, can be considered a safe preservative using cytolytic activity as the means of criteria.

Animals↗
PubMed1984

Sterol carrier protein2 (SCP2)-mediated transfer of cholesterol to mitochondrial inner membranes.

Rats treated with cycloheximide accumulate cholesterol in the adrenal mitochondria. This cholesterol is largely associated with the mitochondrial outer membrane. The addition of homogeneous sterol carrier protein2 (SCP2) to these mitochondria stimulated pregnenolone production to a greater extent than with mitochondria from untreated rats. Adrenal mitochondria from cycloheximide-treated rats were incubated in the presence of aminoglutethimide and cycloheximide to prevent further utilization of cholesterol for pregnenolone synthesis. Outer and inner membrane fractions of these mitochondria incubated with and without 0.75 microM SCP2, were prepared following hypotonic disruption. Incubations of mitochondria in the presence of SCP2 caused a marked shift in cholesterol from outer to inner membrane fractions. The findings provide direct evidence for a role of SCP2 or a similar peptide in modulating transfer of cholesterol to the inner membrane site of cholesterol side chain cleavage.

Adrenal Glands↗
PubMed1983

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization.

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.

Adrenal Glands↗