Biomedical subjects

C M Collins

Publications and source records attributed to C M Collins.

At least 55 records · Page 3Linked to original sources

PubMed1997

Analysis of the interaction between the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) and the human T-cell receptor.

Streptococcus pyogenes that produces the bacterial superantigen streptococcal pyrogenic exotoxin A (SpeA) is associated with outbreaks of streptococcal toxic shock syndrome (STSS) in the United States and Europe. SpeA stimulates V beta 2.1, 12.2, 14.1, and 15.1-positive T cells, and the lymphokine production from the activated T cells is believed to result in the symptoms associated with STSS. The T-cell receptor (TCR)-SpeA interaction is crucial for superantigenic activity, and studies were undertaken to determine regions of both SpeA and the TCR involved in the formation of MHC/SpeA/TCR complexes. Previously, recombinant toxins encoded by speA alleles 1, 2, and 3 as well as toxins resulting from 19 distinct point mutations in speA1 were generated. Here, these 22 toxin forms were incubated with human peripheral blood mononuclear cells (PBMCs), and the percentages of T-cell blasts bearing V beta chains 2.1, 12.2, and 14.1 were quantified by flow cytometry. The analysis indicates that the residues of SpeA needed for a productive TCR interaction differ for each V beta chain examined. An amino acid substitution at only one site significantly affected the toxin's ability to stimulate V beta 2.1-expressing T cells, three individual amino acid substitutions resulted in significant loss of ability to stimulate V beta 12.2-expressing T cells, and substitution at 13 individual sites significantly affected the ability to stimulate V beta 14.1-expressing T cells. To elucidate the regions of the V beta chains that interacted with SpeA, synthetic peptides representative of the human V beta 12.2 complementary-determining regions (CDRs) 1, 2, and 4 were used to block the SpeA-mediated proliferation of human PBMCs. The CDR1, CDR2 and CDR4 peptides were each able to block proliferation, with the activity of CDR1 > CDR2 > CDR4. Combinations of CDR1 peptide with CDR2 or CDR4 peptides allosterically enhanced the ability of each to block proliferation, suggesting SpeA has distinct binding sites for the CDR loops.

Alleles↗

PubMed1996

Three-dimensional mapping of the static magnetic field inside the human head.

Finite element analysis was used to calculate the static magnetic field within the three-dimensional head model. Localized field distributions were evaluated by using the magnetic field histogram technique. Experimental field maps and histograms of the human head were also obtained to validate the simulation results. Field deviations and gradients inside the human head cause NMR signal frequency shifts and line broadening, respectively. Voxels 2 x 2 x 0.5 cm may have frequency differences of more than 2.0 ppm. The linewidth of a single voxel may be broadened by more than 0.5 ppm. Calculated and experimental field maps are in excellent agreement. The global field distortion in the human head is primarily due to the susceptibility difference between air and tissues and their corresponding geometrical shapes.

Computer Simulation↗

PubMed1996

Activation of transcription at divergent urea-dependent promoters by the urease gene regulator UreR.

The Proteus mirabilis and plasmid-encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC-like transcriptional activator. Previously, it was shown that the plasmid-encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea- and UreR-dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid-encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid-encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5 alpha background, as determined by primer-extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR-binding sites within the plasmid-encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid-encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid-encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea.

Bacterial Proteins↗

PubMed1996

Temperature regulation of the streptococcal pyrogenic exotoxin A-encoding gene (speA).

The gene encoding the bacterial superantigen streptococcal pyrogenic exotoxin A is often found in streptococcal strains associated with the recently described streptococcal toxic shock syndrome. Here we demonstrate that this gene is expressed at approximate fourfold higher levels in cells grown at 37 degrees C when compared to cells grown at 26 degrees C. This suggests there is increased production of this toxin when Streptococcus pyogenes is found in infections of the soft tissues and bloodstream, as opposed to S. pyogenes that have not breached the epithelial layers and are living on the surface of the skin.

Exotoxins↗

PubMed1996

Analysis of the superantigenic activity of mutant and allelic forms of streptococcal pyrogenic exotoxin A.

Infections with Streptococcus pyogenes (group A streptococcus) can result in the recently described streptococcal toxic shock syndrome (STSS), which is characterized by rashes, hypotension, multiorgan failure, and a high mortality rate. S. pyogenes isolates associated with STSS usually produce streptococcal pyrogenic exotoxin A (SpeA), a bacterial superantigen capable of stimulating host immune cells. Most of the symptoms of STSS are believed to result from cytokine release by the stimulated cells. To better understand the pathogenesis of STSS, we began studies on the SpeA-immune cell interaction. We generated 20 mutant forms of SpeA1 (SpeA encoded by allele 1), and the mutant toxins were analyzed for mitogenic stimulation of human peripheral blood mononuclear cells, affinity for class II major histocompatibility complex molecules (DQ), and disulfide bond formation. Residues necessary for each of these functions were identified. There are four alleles of speA, and STSS strains usually contain either allele 2 or allele 3. The product of allele 2, SpeA2, had slightly higher affinity for the class II MHC molecule compared with SpeA1 but not significantly greater mitogenic activity. SpeA3, however, was significantly increased in mitogenic activity and affinity for class II MHC compared with SpeA1. Thus, we have evidence that the toxin encoded by some of the highly virulent S. pyogenes STSS-associated isolates is a more active form of SpeA.

Alleles↗

PubMed1996

Identification of a fibronectin-binding protein (GfbA) in pathogenic group G streptococci.

Attachment to eukaryotic cell surfaces is an essential step in the establishment of colonization and infection by bacterial pathogens. This report examines the adherence capabilities of pathogenic group G streptococci and demonstrates that certain group G streptococcal clinical isolates express a fibronectin-binding protein. This protein, termed GfbA for group G streptococcal fibronectin-binding protein, mediates adherence to human skin fibroblasts (HSF). The gene encoding this protein, gfbA, was isolated, and the complete DNA sequence of gfbA was determined. From this sequence GfbA was predicted to be a 580-amino-acid protein (molecular weight = 64,979) with significant amino acid identity to the group A streptococcal fibronectin-binding proteins SfbI and protein F (PrtF) (76 and 78% identity, respectively). GfbA contains regions with notable identity to the fibronectin-binding repeat domains of PrtF. gfbA(+) strains were able to bind to HSF, and preincubation of the gfbA(+) strains with fibronectin blocked this adherence. In addition, gfbA(+) strains were able to bind radiolabeled fibronectin, and this binding was inhibited with addition of excess unlabeled fibronectin. gfbA-negative strains were not able to bind either the HSF or radiolabeled fibronectin. DNA homologous to gfbA was found in 36% of the group G streptococcal isolates examined. Since not all group G streptococcal strains examined contained gfbA, this suggests there might be other tissue-specific adherence molecules expressed by these pathogenic strains.

Adhesins, Bacterial↗

PubMed1996

M proteins of group C streptococci isolated from patients with acute pharyngitis.

We studied 15 strains of group C (Streptococcus equi subsp. equisimilis) [corrected] isolated from the throats of college students with acute pharyngitis and 5 strains isolated from patients with noninfectious problems. Nineteen of the 20 strains resisted phagocytic killing during incubation in normal human blood, suggesting that they might express M proteins. Genomic DNA from all 20 strains hybridized with a probe corresponding to the carboxyterminal one-third of the group A M-protein gene emm24, a region that is highly conserved among M proteins of group A and group G streptococci. The DNA sequences of the N-terminal (variable) regions of the M-protein-encoding genes from two disease-associated group C isolates and one control isolate were determined. The predicted amino acid sequences of the two pharyngitis strains were identical and were 88% homologous to the amino acid sequence of a group G M-protein gene. The predicted terminal amino acid sequence of the control strain does not correspond to any such sequences in the GenBank database. All three strains studied possess the conserved region domain common to class I group A M-protein types epidemiologically associated with rheumatic fever. These studies demonstrate the presence of M proteins in strains of S. equi subsp. equisimilis [corrected] isolated in cases of endemically occurring acute pharyngitis. Certain of these proteins are similar to those of group G streptococci, while others may represent new M types. The similarity in structure and function between M proteins of nonrheumatogenic serogroups and those of rheumatogenic group A streptococci suggests that factors other than or in addition to M protein per se are likely involved in the pathogenesis of rheumatic fever.

Acute Disease↗

PubMed1996

Postmortem diagnosis of Factor V Leiden from paraffin wax embedded tissue.

Activated protein C resistance resulting from Factor V Leiden is an important inherited thrombophilia disorder which is found in 3.5% of people in the UK. The genetic defect can be detected using the PCR and the diagnosis can be made postmortem from paraffin wax embedded tissue. The presence of Factor V Leiden should be sought in all cases of unexplained sudden death resulting from venous thromboembolism.

Journal Article↗

PubMed1996

Weathering nodules of the ear: a clinicopathological study.

Twenty-five cases of distinctive ear nodules on the helices of the ears in elderly males are presented. We believe the clinicopathological features of these lesions allow them to be defined as a distinct entity. They are probably more common than is currently recognized. Histologically, a typical nodule comprises a spur of fibrous tissue with a focus of cartilage metaplasia. While the pathogenesis of weathering nodules remains unclear, there is an association with age and chronic UV exposure.

Adult↗

PubMed1995

UreR activates transcription at multiple promoters within the plasmid-encoded urease locus of the Enterobacteriaceae.

Urease activity is produced by members of the family Enterobacteriaceae that contain the plasmid-encoded urease locus only when urea is present in the growth medium. The plasmid-encoded urease locus contains seven tandem urease structural and accessory genes (ureDABCEFG). Previously we showed that transcription of the first gene in this cluster, ureD, is initiated at a urea-dependent promoter (ureDp). Expression from ureDp requires the product of ureR, which is transcribed divergently from the plasmid-encoded ureDABCEFG. From DNA sequence analysis, UreR is predicted to be a 34 kDa protein with identity to the AraC family of transcriptional activators. In this report we demonstrate that there are two additional urea and UreR-dependent promoters within the plasmid-encoded urease locus: ureRp and ureGp. A low-level constitutive promoter was also identified upstream of ureE (ureEp). Three major mRNA transcripts were induced when urea was present in the growth medium: a transcript containing ureDABCEF, a transcript corresponding to ureG, and a transcript corresponding to ureR. These results indicate that expression of each of the plasmid-encoded urease genes is transcriptionally regulated in response to urea and suggest that there is autogenous regulation of ureR. Therefore UreR is one of three AraC family members described thus far that are positively auto-regulated.

Blotting, Northern↗

PubMed1995

Absence of Epstein-Barr virus in testicular germ cell tumours: a study of 21 cases using in situ hybridisation.

Aim-To establish whether testicular germ cell tumours contain Epstein-Barr virus (EBV) and if so to provide further evidence for the hypothesis that EBV plays a direct role in the pathogenesis of testicular germ cell tumours.Method-Paraffin wax embedded tissue blocks from 21 germ cell tumours including 12 teratomas and nine classic seminomas were examined by in situ hybridisation for the expression of the small EBV encoded nuclear RNA transcripts (EBER-1 and -2) using isotopic and nonisotopic probes.Results-There was no EBER specific signal detectable in any of the testicular germ cell tumours examined by in situ hybridisation whilst a strong signal was observed in appropriate control sections.Conclusion-The absence of demonstrable EBER transcripts in testicular germ cell tumours make a direct role for EBV in the pathogenesis of these tumours unlikely. Other explanations for the epidemiological and serological evidence linking EBV with germ cell tumours need to be explored.

Journal Article↗

PubMed1994

Pediatric renal transplantation in Laurence-Moon-Biedl syndrome.

Two cases of renal transplantation in pediatric patients with Laurence-Moon-Biedl syndrome are reported. Immunosuppressive therapy consisted of cyclosporine, prednisone and azathioprine. Renal function has been good but both patients developed morbid obesity.

Adolescent↗

PubMed1993

Expression of major histocompatibility complex (MHC) antigens and their loss on culture in renal carcinoma.

Biopsies from the tumour and the adjacent normal kidney were obtained from 15 patients with renal cell carcinoma (RCC). The proximal convoluted tubules from which the tumour arose expressed major histocompatibility complex (MHC) class I antigen (Ag) in 3 cases and class II in none. By contrast, the carcinoma cells expressed class I Ag in 14 cases and class II Ag in 5 cases. Cells from each carcinoma were established in culture. As the culture period increased, cells from six of eight RCC showed diminished expression of class I Ag and five of six reduced expression of class II Ag. This is similar to the relative loss of class I Ag in synchronous metastases from RCC.

Carcinoma, Renal Cell↗

PubMed1993

Verapamil enhances doxorubicin activity in cultured human renal carcinoma cells.

Cells from 22 renal cell carcinomas (RCC) were established in culture. Sensitivity of the tumour cells to doxorubicin alone and in combination with racemic verapamil, which reverses multidrug resistance, was tested using a [75Se]selenomethionine uptake assay to measure protein synthesis. The effect of verapamil was expressed as a potentiation index: LD50doxorubicin/LD50doxorubicin + verapamil. The potentiation index in 15 of these carcinomas was determined for cells within the first 14 days of culture. At 3.3 mumol/l concentration of verapamil, of the tumours sensitive to doxorubicin alone (LD50 < 0.75 microgram/ml) five of seven showed a potentiation index of > 2. For the less sensitive tumours the analogous proportion was seven of eight. Tumour cell expression of glycoprotein P-170, associated with multidrug resistance, was estimated using the monoclonal antibody C-219. Initial expression levels were unrelated to the action of verapamil. In five tumours the proportion of cells expressing P-170 declined as the period of culture increased. This was not associated with any consistent change in the LD50 for doxorubicin or in potentiation of doxorubicin sensitivity by verapamil. Cell cloning associated with prolonged cell growth in vitro could mimic tumour cell cloning which accompanies the formation of metastases. Thus reduced expression of P-170 on prolonged cell growth in vitro may be a pointer to the efficacy of combination therapy in the treatment of patients with metastatic renal cell carcinoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

The effect of alpha and gamma interferon on cell growth and histocompatibility antigen expression by human renal carcinoma cells in vitro.

Tumour cells were separated from 19 renal carcinomas and cultured in vitro. The effect of interferon (IFN) alpha and gamma on cell proliferation was measured and compared to the effect of IFN on the expression of class I and class II major histocompatibility complex (MHC) antigens. When tested within the first 14 days of culture, IFN-alpha inhibited protein synthesis in 12 of 15 and IFN-gamma in four of nine tumours. Reduction in cell counts was in parallel. In six tumours the culture period was extended and in all six the effect of IFN-alpha was lost. Exposure to IFN-alpha induced or enhanced class I antigen expression in eight of 19 tumours and class II expression in two of 19. The analogous figures for IFN-gamma were five and three tumours. In four of five cases where a comparison could be made there was a correlation between the effects of IFN-alpha on cell proliferation and class I antigen expression. The efficacy of IFN in the treatment of renal carcinomas may thus, in part, result from inhibition of cell proliferation and enhancement of antigen expression.

Carcinoma, Renal Cell↗

PubMed1993

Identification of a nitrogen-regulated promoter controlling expression of Klebsiella pneumoniae urease genes.

Synthesis of urease by Klebsiella species is known to be induced when the nitrogen source of the growth medium is limiting, suggesting that urease gene expression is controlled by the nitrogen regulatory (ntr) system. This study showed that K. pneumoniae with mutations in either ntrA or ntrC, two integral components of the ntr system, were phenotypically urease-negative. These mutants could be complemented back to a urease positive phenotype with recombinant plasmids encoding the corresponding ntr gene. A series of ure-lacZYA transcriptional fusions, in conjunction with primer extension analysis, identified a DNA region that encoded a nitrogen-regulated promoter. This promoter region controlled transcription of ureD, the first gene in the Klebsiella pneumoniae urease gene cluster, and ureA, a gene that resides immediately downstream of ureD. A high level of transcription from the ureD promoter required NAC, a recently characterized member of the nitrogen regulatory cascade. NAC is a Lys R-like transcriptional regulator that can act at sigma 70 promoters; expression from nac itself is dependent upon NTRA. Therefore, expression of K. pneumoniae urease was dependent upon the nitrogen regulatory cascade, and transcription of at least two urease genes was from a promoter that was positively regulated by NAC.

Amino Acid Sequence↗

PubMed1993

Bacterial ureases: structure, regulation of expression and role in pathogenesis.

The nickel metalloenzyme urease catalyses the hydrolysis of urea to ammonia and carbamate, and thus generates the preferred nitrogen source of many organisms. When produced by bacterial pathogens in either the urinary tract or the gastroduodenal region, urease acts as a virulence factor. At both sites of infection urease is known to enhance the survival of the infecting bacteria. Ammonia resulting from the action of urease is believed to increase the pH of the environment to one more favourable for growth, and to injure the surrounding epithelial cells. In addition, in the urinary tract urease activity can result in the formation of urinary calculi. Bacterial urease gene clusters contain from seven to nine genes depending upon the species. These genes encode the urease structural subunits and accessory polypeptides involved in the biosynthesis of the nickel metallocentre. So far, three distinct mechanisms of urease gene expression have been described for ureolytic bacteria. Some species constitutively produce urease; some species produce urease only if urea is present in the growth medium; and some species produce urease only during nitrogen-limiting growth conditions. For either the urea-inducible genes or the nitrogen-regulated genes transcription appears to be positively regulated. In the nitrogen-regulated systems, urease gene expression requires Nac (nitrogen assimilation control), a member of the LysR family of transcriptional activators. Urea dependent expression of urease requires UreR (urease regulator), a member of the AraC family of transcriptional activators. An evolutionary tree for urease genes of eight bacterial species is proposed.

Bacteria↗

PubMed1993

The plasmid-encoded urease gene cluster of the family Enterobacteriaceae is positively regulated by UreR, a member of the AraC family of transcriptional activators.

Ureolytic clinical isolates of Providencia stuartii, Salmonella spp., and some Escherichia coli strains contain large urease-encoding plasmids. Expression of urease activity from these isolates is induced at least 20-fold by urea. In order to facilitate studies on the regulatory mechanism controlling this urea-inducible expression, the plasmid-encoded urease genes were inserted into the low-copy-number vector pRK415, to form pSEF70. Deletion mutagenesis of pSEF70 demonstrated that between 1.3 and 1.6 kb of DNA upstream of ureD (the first of seven urease genes clustered in an operon-like fashion) was required for a urease-positive phenotype. An open reading frame coding for a 34.1-kDa polypeptide was found in the DNA sequence of this upstream region. This open reading frame has been designated ureR, for urease regulator. A urea-inducible promoter region was identified upstream of ureD. Transcription from this promoter was activated only when ureR was present in trans. The predicted ureR gene product contains a helix-turn-helix motif and shows significant amino acid similarity to the AraC family of transcriptional activators. We conclude that urea-dependent expression from the plasmid-encoded urease gene cluster requires ureR and that ureR codes for a positive regulatory element controlling transcription of at least one essential urease gene, ureD.

Amino Acid Sequence↗