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Biomedical subjects

T Fowler

Publications and source records attributed to T Fowler.

At least 19 recordsLinked to original sources
PubMed2001

Fibronectin binding protein A of Staphylococcus aureus can mediate human T lymphocyte adhesion and coactivation.

The extracellular matrix protein fibronectin (FN) mediates the adhesion of bacteria as well as T lymphocytes. Mammalian cells express integrins alpha(4)beta(1) and alpha(5)beta(1) as the major FN-binding cell surface receptors. Bacteria such as Staphylococcus aureus, also express FN-binding receptors that are important for adherence to host tissue and initiation of infection. The S. aureus FN-binding protein, FnbpA, has been previously identified, and recombinant proteins that correspond to distinct functional regions of this protein have been made. Three recombinant truncated forms of FnbpA, rFnbpA(37-881), rFnbpA(37-605), and rFnbpA(620-881), were examined for effects on in vitro adhesion and coactivation of human T lymphocytes. These proteins, when coimmobilized with anti-CD3 mAb, activated T lymphocyte proliferation. The coactivation signal generated by the rFnbpA proteins required medium containing serum with FN. Furthermore, the costimulatory signal could be restored in FN-depleted serum when the rFnbpAs were preloaded with soluble FN. Monoclonal Ab blocking studies revealed that integrin alpha(5)beta(1) is the major receptor responsible for the rFnbpA costimulatory signal. Shear flow cell detachment assays confirmed that lymphocytes can bind to FN captured by the rFnbpA proteins. These results suggest that the S. aureus rFnbpA can interact with integrin alpha(5)beta(1) via an FN bridge to mediate adhesion and costimulatory signals to T lymphocytes.

Adhesins, Bacterial↗
PubMed2001

Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells.

Invasive Staphylococcus aureus infection frequently involves bacterial seeding from the bloodstream to other body tissues, a process necessarily involving interactions between circulating bacteria and vascular endothelial cells. Staphylococcus aureus fibronectin-binding protein is central to the invasion of endothelium, fibronectin forming a bridge between bacterial fibronectin-binding proteins and host cell receptors. To dissect further the mechanisms of invasion of endothelial cells by S. aureus, a series of truncated FnBPA proteins that lacked one or more of the A, B, C or D regions were expressed on the surface of S. aureus and tested in fibronectin adhesion, endothelial cell adhesion and invasion assays. We found that this protein has multiple, substituting, fibronectin-binding regions, each capable of conferring both adherence to fibronectin and endothelial cells, and endothelial cell invasion. By expressing S. aureus FnBPA on the surface of the non-invasive Gram-positive organism Lactococcus lactis, we have found that no other bacterial factor is required for invasion. Furthermore, we have demonstrated that, as with other cell types, invasion of endothelial cells is mediated by integrin alpha5beta1. These findings may be of relevance to the development of preventive measures against systemic infection, and bacterial spread in the bacteraemic patient.

Adhesins, Bacterial↗
PubMed2001

The current status of maggot therapy in wound healing.

The clinical use of sterile maggots under the brand name LarveE has increased steadily in the UK since they were introduced in the UK in late 1995. Some 50 articles have been published in the last 4 years that describe the cost-effectiveness of maggot therapy in a variety of wound types including leg ulcers, pressure ulcers and diabetic ulcers. The ability of these creatures to combat wound infection has been well documented including those caused by antibiotic-resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus. No significant risks or adverse events have been linked to the use of maggots, although their presence may increase pain levels in leg ulcers associated with ischaemic disease.

Animals↗
PubMed2000

Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins.

Although Staphylococcus aureus is primarily considered an extracellular pathogen, recent evidence suggests that this bacterium can invade a variety of nonprofessional phagocytic cells. Here we investigate the early stages of cellular invasion by S. aureus and determine the bacterial and host components that are required for this process. S. aureus expresses two cell surface-associated fibronectin (FN)-binding proteins (FnbpA and FnbpB) that mediate the interaction of the bacteria with both soluble and solid-phase FN in vitro. Using a mutant of S. aureus that lacks the expression of both Fnbps, we show that the expression of either protein is necessary for efficient uptake by the mouse fibroblast line GD25beta1A. Invasion could be inhibited by soluble recombinant proteins encompassing either the FN-binding D repeat region or the A region (and B repeats) of FnbpA, suggesting that the activities of both regions are important in this process. We demonstrate that FN is also required for invasion of this cell line. In the presence of FN-depleted fetal bovine serum, the invasion level was reduced by approximately 40% compared to in the presence of whole fetal bovine serum. Invasion could be further reduced by the addition of anti-mouse FN antibodies to the assay. Finally, we utilize a mutant mouse fibroblast line, which lacks beta1 integrin expression, to demonstrate that host cell beta1 integrins are necessary for efficient cellular invasion. The level of invasion of the mutant cell line GD25 was reduced by approximately 97% compared to the beta1-expressing complemented cell line GD25beta1A. In addition, invasion of the GD25beta1A cell line could be inhibited by an RGD-containing peptide, further implicating a role for integrins in this process. Based on these observations, we put forward a model of S. aureus invasion in which host FN forms a bridge between the bacterial Fnbps and host cell beta1 integrins, leading to bacterial uptake.

Adhesins, Bacterial↗
PubMed1998

Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss.

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with the type of autosomal recessive nonsyndromic neurosensory deafness known as "DFNB1." Studies indicate that DFNB1 (13q11-12) causes 20% of all childhood deafness and may have a carrier rate as high as 2. 8%. This study describes the analysis of 58 multiplex families each having at least two affected children diagnosed with autosomal recessive nonsyndromic deafness. Twenty of the 58 families were observed to have mutations in both alleles of Cx26. Thirty-three of 116 chromosomes contained a 30delG allele, for a frequency of .284. This mutation was observed in 2 of 192 control chromosomes, for an estimated gene frequency of .01+/-.007. The homozygous frequency of the 30delG allele is then estimated at .0001, or 1/10,000. Given that the frequency of all childhood hearing impairment is 1/1,000 and that half of that is genetic, the specific mutation 30delG is responsible for 10% of all childhood hearing loss and for 20% of all childhood hereditary hearing loss. Six novel mutations were also observed in the affected population. The deletions detected cause frameshifts that would severely disrupt the protein structure. Three novel missense mutations, Val84Met, Val95Met, and Ser113Pro, were observed. The missense mutation 101T-->C has been reported to be a dominant allele of DFNA3, a dominant nonsyndromic hearing loss. Data further supporting the finding that this mutation does not cause dominant hearing loss are presented. This allele was found in a recessive family segregating independently from the hearing-loss phenotype and in 3 of 192 control chromosomes. These results indicate that 101T-->C is not sufficient to cause hearing loss.

Alleles↗
PubMed1998

Written lists as mediating stimuli in the matching-to-sample performances of individuals with mental retardation.

Students with mental retardation learned to write lists in order to perform a matching task that they could not do otherwise. After an initial assessment phase, reinforcement was arranged in the computerized tasks to follow selection of the six pictures that were identical to those in the six-picture samples presented. In Study 1, even though the participants wrote a list of the names of the six sample pictures on each trial, read a list, or did both, they often made errors when a brief delay preceded picture selection. In contrast, performance was nearly perfect when a list was written, read, and remained available at the time of picture selection, suggesting that the list served to mediate the delays. Study 2 examined the stimulus control by two- and six-picture samples over the list writing. Early during testing, 1 participant refrained from writing lists on two-picture trials but wrote lists on six-picture trials, thereby maximizing reinforcement and minimizing its delay; the other participant showed this pattern of list writing after supplemental training. The studies suggest methods for establishing a rudimentary repertoire of mediating behavior that has relevance for teaching instruction-following skills in natural settings.

Achievement↗
PubMed1996

Secretion of Trichoderma reesei beta-glucosidase by Saccharomyces cerevisiae.

An intronless form of the bgl1 gene encoding an extracellular beta-glucosidase from Trichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete active beta-glucosidase into the growth medium. Additionally, active recombinant beta-glucosidase protein was shown to be localized predominantly in the periplasmic space by using a p-nitrophenyl beta-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10-15 kDa larger than that of the native form. Treatment of the recombinant beta-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.

Base Sequence↗
PubMed1995

The effect of noise trauma following training exposures in the mouse.

The effect of moderate level acoustic stimulation, or 'training', on a subsequent high intensity noise exposure was studied in CBA/Ca mice. Eight groups of mice were exposed to a variety of training paradigms as well as different intensity traumatic exposures. We sought a combination which would result in the maximum protective effect from acoustic trauma as measured by the auditory brainstem responses. Using a narrow band noise centered at 4.5 kHz, we investigated the effects of a 10-day 'interval' training regimen, allowing a rest period between successive training exposures, as well as several continuous training exposures. These training paradigms were followed by a 24 h traumatic noise exposure (also centered at 4.5 kHz) at one of three intensities, 107, 110, or 117 dB SPL which induce a temporary, a moderate, or a severe permanent threshold shift, respectively. In none of these trained groups was a protective effect demonstrated at any time up to one month following a subsequent traumatic noise exposure. Several groups demonstrated higher compound threshold shifts after the traumatic noise exposure compared to controls. After a recovery period of 4 weeks nearly all trained groups demonstrated a tendency toward higher permanent threshold shifts than the control, untrained, animals. While no protective effect was demonstrated, examination of the threshold shifts following the training periods and after the traumatic noise exposures raised interesting questions for future investigation regarding the inherent resistance to noise induced threshold shifts in the mouse.

Acoustic Stimulation↗
PubMed1993

The catR gene encoding a catalase from Aspergillus niger: primary structure and elevated expression through increased gene copy number and use of a strong promoter.

Synthetic oligonucleotide probes based on amino acid sequence data were used to identify and clone cDNA sequences encoding a catalase (catalase-R) of Aspergillus niger. One cDNA clone was subsequently used to isolate the corresponding genomic DNA sequences (designated catR). Nucleotide sequence analysis of both genomic and cDNA clones suggested that the catR coding region consists of five exons interrupted by four small introns. The deduced amino acid sequence of catalase-R spans 730 residues which show significant homology to both prokaryotic and eukaryotic catalases, particularly in regions involved in catalytic activity and binding of the haem prosthetic group. Increased expression of the catR gene was obtained by transformation of an A. niger host strain with an integrative vector carrying the cloned genomic DNA segment. Several of these transformants produced three- to fivefold higher levels of catalase than the untransformed parent strain. Hybridization analyses indicated that these strains contained multiple copies of catR integrated into the genome. A second expression vector was constructed in which the catR coding region was functionally joined to the promoter and terminator elements of the A. niger glucoamylase (glaA) gene. A. niger transformants containing this vector produced from three- to 10-fold higher levels of catalase-R than the untransformed parent strain.

Amino Acid Sequence↗
PubMed1993

A transcriptional switch between the Pig-1 and Sgs-4 genes of Drosophila melanogaster.

Pig-1 and Sgs-4 are a pair of closely linked and divergently transcribed Drosophila melanogaster genes, which are both expressed in larval salivary glands but at different times during development. While Sgs-4 is expressed at high levels only at the end of the third instar, Pig-1 exhibits a major peak of expression during late second and early third instar. Thus, Pig-1 expression declines as Sgs-4 expression is induced. In this paper, we show that three adjacent elements located within the short region between these genes can account for the switch from Pig-1 to Sgs-4 expression. A 170-bp segment acts as an enhancer to direct Sgs-4 expression in late-third-instar salivary glands. A 64-bp sequence located just upstream from the enhancer can modify its temporal specificity so that it works throughout the third instar. Expression induced at mid-third instar by a combination of these two elements can be repressed by a negative regulatory sequence located still further upstream. We present evidence suggesting that the changing interactions between these regulatory elements and the Sgs-4 and Pig-1 promoters lead to the correct pattern of expression of the two genes.

Age Factors↗
PubMed1992

The bgl1 gene encoding extracellular beta-glucosidase from Trichoderma reesei is required for rapid induction of the cellulase complex.

We have used a targeted gene deletion event to remove the coding region for the bgl1 gene encoding an extracellular beta-glucosidase from the genome of the cellulolytic fungus Trichoderma reesei. The bgl1 null mutants were used to investigate the role of beta-glucosidase in the hydrolysis of cellulose and induction of the other cellulolytic enzyme components. In the absence of extracellular beta-glucosidase, growth of bgl1 null strains on several carbon sources was the same as that of the parent (as measured by mycelial dry weight). However, levels of extracellular protein and total endoglucanase production were seen to lag relative to those levels observed in the control strain. The mRNA levels of the CBHI, CBHII, EGI, and EGII cellulase genes (cbh1, cbh2, egl1 and egl3) showed a corresponding lag in induction, suggesting that the absence of extracellular beta-glucosidase has an effect on the co-ordinate regulation of the other cellulase genes at the level of transcription. The addition of a potent inducer of the cellulase complex (sophorose) resulted in normal rates of cellulase gene mRNA production and extracellular protein release. This indicates that the absence of beta-glucosidase is not affecting some intrinsic cellular ability to produce mRNA or secrete protein. These data suggest that a functional beta-glucosidase is at least partially responsible for the efficient induction of the depolymerase enzymes of the cellulase complex. The observation that the cellulase complex is induced, albeit after a lag, suggests that other enzymes are present that can substitute for the function of beta-glucosidase during induction.

Cellulase↗
PubMed1991

Gene expression systems for filamentous fungi.

The extraordinary capacity of filamentous fungi to produce large quantities of extracellular protein, together with the advent of DNA-mediated fungal transformation, has resulted in rapid advances in the development of gene expression systems for filamentous fungi. This review focuses on recent developments in the expression of both fungal and non-fungal genes and improvements to the host.

Animals↗
PubMed1991

Cloning and amplification of the gene encoding an extracellular beta-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates.

We have cloned and determined the nucleotide sequence of the gene encoding an extracellular beta-glucosidase (bgl1) from the cellulolytic fungus Trichoderma reesei. The predicted open reading frame of the bgl1 gene is interrupted by two putative introns of 70 and 64 bp and encodes a protein with a calculated molecular weight of 75,341. The genomic segment encoding bgl1 was cloned into a vector that contained the selectable marker gene, amdS. Transformation of T. reesei with this vector resulted in several stable transformant strains all possessing an increased copy number of the bgl1 gene integrated into the genome together with elevated rates of glucose production from avicel. One transformant produced an extracellular cellulase with a five-fold increase in the rate of production of glucose from cellobiose, a 33% rate increase from avicel, and a 17% increase from phosphoric acid swollen cellulose. These data suggest that the cellulolytic activity of T. reesei strains may be specifically improved by transformation with cloned cellulase genes.

Amino Acid Sequence↗
PubMed1990

Regulation of the glaA gene of Aspergillus niger.

The glucoamylase gene of Aspergillus niger, glaA, is expressed at high levels in the presence of starch. We have determined the nucleotide sequence of 1966 bp of the 5' flanking region of the glaA gene and have begun to identify sequences important for the control of glaA expression by deletion analysis. Constructs containing deletions extending into the glaA gene promoter were introduced into an A. niger host whose own glaA gene had been disrupted by a gene replacement event. Secreted levels of glucoamylase, expressed from each of the recombinant glaA genes, were measured by enzyme immunoassay. The effect of each deletion on the expression of glaA, when grown on differing carbon sources, was used to determine the limits of sequences upstream of glaA responsible for gene regulation. A region between -562 and -318 appears to direct high-level expression, whereas only 214 bp of 5' flanking sequence is required to initiate the start to transcription.

Amino Acid Sequence↗
PubMed1988

Functional redundancy in the tissue-specific enhancer of the Drosophila Sgs-4 gene.

During the last day of larval development, the Sgs-4 glue gene of Drosophila melanogaster is expressed at high levels in a single tissue, the larval salivary glands. As shown by transformation experiments and by DNA sequence analysis of Sgs-4 underproducing strains, an essential regulatory region for Sgs-4 expression lies between 149 and 568 bp upstream from the transcribed part of the gene. This region shows the positional independence of a transcriptional enhancer and directs at least three regulatory activities: tissue specificity, developmental timing and high-level expression. Here we use a transient transformation assay to identify three elements within this enhancer that are involved in tissue specificity. For at least this regulatory activity the enhancer is internally redundant. Any pairwise combination of the three elements is sufficient to direct salivary gland expression, although none of the three can act alone.

Animals↗
PubMed1986

Shadow promoters in the F plasmid transfer operon.

The in vivo transcription start site for PYZ, the promoter of the F plasmid transfer operon, has been located. When transcription start sites for PYZ are deleted, two additional shadow promoters, upstream of PYZ, are activated in vivo. The shadow promoters correspond to those previously identified by in vitro run-off transcription experiments. The activated shadow promoters are stimulated by TraJ protein, a positive regulator of the transfer operon.

Base Sequence↗
PubMed1983

The control region of the F plasmid transfer operon: DNA sequence of the traJ and traY genes and characterisation of the traY leads to Z promoter.

The complete nucleotide sequence of the F plasmid transfer genes traJ and traY, together with the promoter-proximal region of the traA gene has been determined. The traJ reading frame has been confirmed by sequencing the traJ90 amber mutant allele. The predicted amino acid sequence of the TraJ protein shows that this outer-membrane protein lacks a signal sequence. The pattern of codon usage within the traJ gene is different from that of genes for abundant outer-membrane proteins and is closer to that of genes that are expressed at relatively low levels. We have located the traY leads to Z operon promoter by in vitro run-off transcription experiments and have developed in vivo assays for the activity of the promoter by fusing it to galactokinase and kanamycin-resistance genes.

Bacterial Proteins↗