Biomedical subjects

S Clegg

Publications and source records attributed to S Clegg.

At least 37 records · Page 2Linked to original sources

PubMed1996

Salmonella typhimurium fimbrial phase variation and FimA expression.

Bacteria in a nonfimbriate phase because of continuous aeration of liquid cultures produce FimA in amounts similar to those produced by fimbriate bacteria. However, relatively low FimA production was observed in nonfimbriate-phase cultures obtained by growth on solid media or by anaerobic incubation. Regardless of the fimbrial phase of Salmonella typhimurium, the fimA promoter region was always oriented in the direction that might allow fimA transcription.

Anaerobiosis↗

PubMed1995

Adherence properties of an mrkD-negative mutant of Klebsiella pneumoniae.

The role of the mrkD gene in attachment by a type 3 fimbriate Klebsiella pneumoniae strain was further characterized. A clinical isolate, K. pneumoniae IA565, was found to contain two copies of the gene encoding the fimbrial subunit, mrkA, and one copy of the gene encoding the adhesin subunit, mrkD. One copy of mrkA was located on the bacterial chromosome, and the other copy was associated with mrkD and located on a plasmid. The plasmid-borne mrk gene cluster was lost when K. pneumoniae IA565 was subcultured serially in broth at 44 degrees C. The resulting mrkD-negative strain, designated K. pneumoniae IApc35, did not exhibit the following adherence characteristics associated with K. pneumoniae possessing MrkD-positive fimbriae: agglutination of tannic acid-treated human erythrocytes and attachment to trypsinized human buccal cells. However, K. pneumoniae IApc35 produced type 3 fimbriae that were composed of the characteristic 21.5-kDa major fimbrial subunit, were reactive with specific serum, and were visualized specifically by immunoelectron microscopy. K. pneumoniae IApc35 retained a copy of the mrkA gene on its chromosome. This mrkA-containing gene cluster could be complemented by a recombinant plasmid carrying only the mrkD gene, resulting in restoration of the K. pneumoniae IA565-like adhesive phenotype and demonstration of type 3 filament-associated MrkD subunits by using colloidal gold labeling and immunoelectron microscopy. These data indicate that K. pneumoniae may contain multiple copies of the mrk genes which may be present simultaneously on both plasmid and chromosomal DNAs and which may encode fimbriae with different binding specificities.

Adhesins, Bacterial↗

PubMed1995

Construction and characterization of a fimZ mutant of Salmonella typhimurium.

The Salmonella typhimurium fimA gene is controlled by several ancillary fim genes. One of these genes, fimZ, appears to be involved in increasing the expression of fimA. A fimZ mutant of S. typhimurium was constructed by allelic exchange, and this mutant was found to be nonfimbriate. The fimZ mutant demonstrated decreased levels of fimA expression compared with the parental strain when both were grown under conditions favoring fimbrial expression. An examination of the predicted amino acid sequence, deduced from the nucleotide sequence of fimZ, indicated that the FimZ polypeptide possessed a DNA binding motif. Bacterial lysates, derived from strains transformed with recombinant plasmids possessing a fimZ gene, demonstrated DNA binding activity with a fragment containing the fimA promoter. Lysates without a FimZ polypeptide did not exhibit any binding activity. These data are consistent with FimZ being a transcriptional activator of fimA, and FimZ acts by binding to the promoter region.

Bacterial Proteins↗

PubMed1994

The gene fimU affects expression of Salmonella typhimurium type 1 fimbriae and is related to the Escherichia coli tRNA gene argU.

The gene fimU, located on a recombinant plasmid carrying the Salmonella typhimurium type 1 fimbrial gene cluster is closely related to the Escherichia coli tRNA gene argU. The fimU gene complements an E. coli argU mutant that is a P2 lysogen, thereby allowing the phage P4 to grow in this strain but preventing the growth of phage lambda. In addition, fimU was shown to be involved in fimbrial expression since transformants of the E. coli argU mutant could produce fimbriae only in the presence of fimU but not in its absence, whereas in an E. coli argU+ strain fimbriation did not require the fimU gene.

Agglutination↗

PubMed1994

Heating rate modeling and measurements in phantom and in vivo of the human upper extremity in a defective 2450 MHz microwave oven.

Multiple door interlock switches in commercial microwave ovens are designed to prevent accidental exposure and injury. We report a) heating rate (degree/sec) measurements in a phantom of the human upper extremity in a 2450 MHz microwave oven having interlock switches deliberately bypassed; b) skin temperature measurements on the upper extremity of a human volunteer similarly exposed; c) perception of warmth and pain experienced by the volunteer during exposure; d) thermographic camera recordings of the volunteer's skin; and e) finite element modeling of specific absorption rate (SAR) in the volunteer's hand. Moderately severe pain was experienced at the fingertips after 5 sec of exposure, consistent with the modeled SAR, measured heating rates, and published data on the temperature threshold for pain. We estimate that an additional 9 sec of exposure would be required to produce irreversible injury, consisting of focal thermal injury in the fingertips and possibly the thenar and hypothenar eminences.

Computer Simulation↗

PubMed1994

Relationship between chromosome aberrations, micronuclei and cell kill in two human tumour cell lines of widely differing radiosensitivity.

The relationship between chromosome aberration frequency, micronucleus (MN) formation and cell kill was investigated in two human tumour cell lines, RT112, a radioresistant bladder carcinoma cell line, and HX142, a radiosensitive neuroblastoma cell line. As MN are generally accepted to result from acentric chromosome fragments that are lost at nuclear division and all unstable aberrations result in an acentric fragment, a 1:1 correlation between aberrations and MN might be expected. There was a good correlation between cell kill and aberration frequency in the two cell lines, however the relationship between cell kill and MN formation in the lines was very different. RT112 showed 0.22 aberrations and 0.21 MN per Gy whilst HX142 showed 0.61 aberrations and 0.078 MN per Gy. Possible reasons for the discrepancy observed in HX142 are discussed.

Cell Survival↗

PubMed1994

The type 3 fimbrial adhesin gene (mrkD) of Klebsiella species is not conserved among all fimbriate strains.

The type 3 fimbriae of enteric bacteria mediate agglutination, in vitro, of erythrocytes treated with tannic acid. The gene encoding the polypeptide, MrkD, that mediates this agglutination reaction was placed downstream of an inducible promoter, and the ability of MrkD alone to facilitate hemagglutination was determined. Although Escherichia coli transformants could be shown to produce the MrkD protein, hemagglutination did not occur in the absence of other mrk gene products. In addition, the MrkD polypeptide did not cross the bacterial outer membrane unless a fimbrial chaperone protein was also present. Analysis of the frequency of the mrkD gene within the genus Klebsiella indicated that this gene is conserved in strains of Klebsiella oxytoca but not in other fimbriate Klebsiella species. In the small number of strains of Klebsiella pneumoniae that do possess a related mrkD gene, this determinant could be found on a plasmid in one strain. The ability of type 3 fimbriate bacteria to adhere to type V collagen was found to be a function of a specific MrkD polypeptide. This adhesin is frequently found in strains of K. oxytoca but is rarely associated with the type 3 fimbriae of K. pneumoniae.

Adhesins, Bacterial↗

PubMed1994

Type 1 fimbrial shafts of Escherichia coli and Klebsiella pneumoniae influence sugar-binding specificities of their FimH adhesins.

The type 1 fimbriae of enterobacteria comprise FimA, which constitutes most of the fimbrial shaft, and a cassette of three minor ancillary subunits including FimH, the mannose-binding moiety. The sugar-binding specificities of Escherichia coli and Klebsiella pneumoniae type 1 fimbriae were examined by determining the relative activities of two aromatic mannosides in inhibiting the yeast aggregation caused by the fimbriated bacteria. 4-Methylumbelliferyl alpha-mannoside (MeUmb alpha Man) was approximately 10-fold more effective than p-nitrophenyl alpha-mannoside (p-NP alpha Man) in inhibiting the yeast aggregation caused by the recombinant expressing native E. coli type 1 fimbriae. In contrast, MeUmb alpha Man was only fourfold more effective than p-NP alpha Man in assays employing the recombinant expressing native K. pneumoniae type 1 fimbriae. In order to elucidate the molecular mechanisms underlying the sugar-binding specificities of type 1 fimbriae in the two species, transcomplementation studies were performed and resulted in the creation of recombinants expressing two types of hybrid fimbriae: one consisting of a cassette of minor subunits of E. coli fimbriae borne on a filamentous shaft of K. pneumoniae FimA subunits and the other consisting of a cassette of K. pneumoniae minor fimbrial subunits borne on a shaft of E. coli FimA subunits. Although the heterologous FimH was incorporated into the fimbrial filaments in amounts comparable to those observed in native fimbriae, the hemagglutination activities of recombinants expressing hybrid fimbriae were significantly lower than those of their counterparts bearing native fimbriae. The sugar-binding specificity of the recombinant expressing hybrid fimbriae consisting of an E. coli shaft bearing K. pneumoniae FimH was different from those of recombinants expressing native K. pneumoniae fimbriae in its affinity for the two aromatic sugars but was remarkably similar to the specificities exhibited by recombinants expressing native E. coli fimbriae. Conversely, the sugar-binding specificity of the recombinant expressing hybrid fimbriae consisting of a K. pneumoniae shaft bearing E. coli FimH was different from that of the recombinant expressing native E. coli fimbriae but was very similar to those of recombinants expressing native K. pneumoniae fimbriae. We conclude that the differences in the sugar-binding specificity between E. coli and K. pneumoniae FimH fimbrial subunits is influenced by the fimbrial shafts which carry the adhesin molecules in a functionally competent form at the distal tips.

Adhesins, Escherichia coli↗

PubMed1993

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin.

Adherence of type-1-fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type-1-fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non-fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside-inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(pISF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae-coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type-1-fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type-1-fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type-1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(pISF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type-1 fimbriae of S. enterica and E. coli bind to oligomannoside chains of the laminin network in basement membranes.

Adhesins, Escherichia coli↗

PubMed1992

Relationships among tumor temperature, treatment time, and histopathological outcome using preoperative hyperthermia with radiation in soft tissue sarcomas.

The lack of an unambiguous thermal dosimetry continues to impede progress in clinical hyperthermia. In an attempt to define better this dosimetry, a model based on the cumulative minutes during which arbitrary percentages of measured tumor temperature points exceeded an index temperature was tested in patients with soft tissue sarcomas treated with preoperative hyperthermia and conventional radiation therapy. Patients received 5000-5040 cGy at 180-200 cGy per fraction. Hyperthermia was delivered 30-60 minutes after radiation therapy and given for 60 minutes. Patients were randomized between one and two hyperthermia treatments per week for a total of five or 10 treatments, respectively. Lesions were excised 4-6 weeks after completion of hyperthermia/radiation therapy. Successful treatment outcome was considered to be the finding of greater than 80% necrosis of the sarcoma upon histopathologic examination of the resected specimen. Forty-five patients were eligible with thermometry data available in 44 patients. An average of 19 interstitial sites were monitored each treatment per tumor. Sixty percent of tumors had a successful histopathologic outcome. Univariate analysis demonstrated that several descriptors of the temperature distribution were strongly related to treatment outcome; more strongly than nonthermometric factors, such as the number of treatments per week, tumor volume and patient age and more strongly than the commonly used temperature descriptors Tmin and Tmax. Descriptors that incorporated both temperature and time were also superior to the more commonly used descriptors Tmin and Tmax. Multivariate stepwise logistic regression analysis revealed that a descriptor of both the hyperthermia treatment time and the frequency distribution of intratumoral temperatures was the strongest predictor of histopathologic outcome and that the best predictive model combined this time/temperature descriptor and one versus two treatment per week grouping. The more conventional temperature descriptor, minimum measured tumor temperature, did not significantly enhance the predictive power of treatment group. Based on these results, we recommend that descriptors based on both the frequency distribution of intratumoral temperatures and hyperthermia treatment time be tested for relationships with treatment outcome in other clinical data bases. Furthermore, we recommend that temperature descriptors that are less sensitive to catheter placement and tumor boundary identification than Tmin and Tmax (such as T90, T50, and T10) be tested prospectively along with other important thermal variables in Phase II trials in further efforts to define a thermal dosimetry for spatially nonuniform temperature distributions.

Combined Modality Therapy↗

PubMed1992

Fimbriation, capsulation, and iron-scavenging systems of Klebsiella strains associated with human urinary tract infection.

Thirty-two strains of Klebsiella pneumoniae and seven strains of Klebsiella oxytoca isolated from urinary tract infections in elderly adults were analyzed for capsular antigens, iron-scavenging systems, and fimbriation. All strains were capsulated. Twenty-seven different K antigens were identified among the strains, with no particular antigen dominating. All strains produced the iron-scavenging system enterochelin as analyzed by bioassay and DNA hybridization. In contrast, the aerobactin iron-sequestering system was not detected in any of the strains. All strains caused hemagglutination of tannin-treated human erythrocytes and reacted with an anti-type 3 fimbriae antiserum as well as in DNA hybridization with a type 3 fimbria-specific probe, indicating that the Klebsiella strains possessed this fimbrial type. Possession of type 1 fimbriae was analyzed by agglutination tests and by hybridization with DNA probes from two distinct Klebsiella type 1 fimbria gene clusters. Phenotypic expression of the type 1 fimbriae was found in 29 of 32 K. pneumoniae strains, whereas 30 strains reacted with either of the two type 1 fimbrial cluster DNA probes. In K. oxytoca, however, only three of seven strains expressed type 1 fimbriae and reacted with the DNA probes. The type 3 fimbriae were found to bind to a fraction of epithelial cells exfoliated in normal human urine, whereas the type 1 fimbriae bound strongly to urinary slime. No inhibitors of type 3 fimbrial binding were detected in human urine.

Aged↗

PubMed1992

Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products.

We examined the role of Klebsiella fimbrial types 1 and 3 in mediating adherence to human buccal and tracheal cells and to lung tissue sections. We found that clinical isolates of Klebsiella pneumoniae producing type 3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression of Klebsiella type 3 fimbriae (pFK10) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissue sections, in contrast to nonfimbriate and to type 1 fimbriate bacteria. Adherence by type 3 fimbriate bacteria was inhibited by purified type 3 fimbriae and Fab fragments derived from type 3 fimbrial-specific polyclonal immunoglobulin G. Type 3 fimbriae mediated attachment to the basolateral surface of tracheal cells and to the basal epithelial cells and the basement membrane regions of bronchial epithelia. Using an E. coli transformant (pDC17/pFK52), which expresses nonadherent P fimbrial filaments, along with the type 3 fimbrial adhesin (MrkD), we demonstrated that type 3 fimbrial attachment to respiratory cells was attributable to the MrkD adhesin subunit. Subsequent experiments demonstrated that the epithelial target of the type 3 fimbrial adhesin was most likely a peptide molecule rather than a carbohydrate. The results of this study demonstrate that, in vitro, the Klebsiella type 3 fimbrial adhesin mediates adherence to human respiratory tissue.

Acetates↗

PubMed1992

Identification of ancillary fim genes affecting fimA expression in Salmonella typhimurium.

Regulation of the gene, fimA, encoding the major fimbrial subunit of S. typhimurium S6704 was examined by using a lambda fimA-lacZ lysogen. Transformation of the lambda fimA-lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression, pISF101, indicated that two regions of this plasmid alter beta-galactosidase production. One plasmid is a deletion resulting in the loss of a 28-kDa polypeptide downstream of fimA, while the other plasmid encodes a 24- and a 27-kDa polypeptide. Northern (RNA) blot analyses indicated that the steady-state fimA mRNA levels of these transformants were high. In addition, phenotypic expression of type 1 fimbriae by agar-grown cultures is observed only in those transformants bearing plasmids which show increased beta-galactosidase and fimA mRNA levels.

Amino Acid Sequence↗

PubMed1991

The frequency of fim genes among Salmonella serovars.

Salmonella serovars were examined for the presence of fim gene sequences using specific DNA probes. All strains, regardless of their ability to express surface-associated fimbriae, retain a considerable amount of DNA homologous to the gene probes used. The phenotypically nonfimbriae FIRN and non-FIRN strains of S. typhimurium retain detectable amounts of fim gene sequences and, therefore, may not be genotypically non-fimbriate. The MS adhesin can be expressed by type 2 fimbriate bacteria when they are transformed with discrete regions of the fim gene cluster. However, this conversion to a hemagglutinating phenotype is not associated with a small region of DNA. Therefore, the inability of type 2 fimbria-producing strains of Salmonella to mediate hemagglutination does not appear to be due to a small deletion in a single fim gene.

Adhesins, Bacterial↗

PubMed1991

Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae.

The nucleotide sequence of six genes involved in the expression of type 3 fimbriae of Klebsiella pneumoniae was determined. In addition to the genes that encode the fimbrial subunit (mrkA) and adhesion (mrkD), the mrkB, mrkC, and mrkE genes appear to be involved in assembly of the fimbrial filament and regulation of type 3 fimbrial expression. The mrkF gene product is required to maintain the stability of the fimbrial filament on the cell surface.

Adhesins, Bacterial↗

PubMed1991

Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae.

Bacterial attachment is believed to be an early step in gram-negative nosocomial pneumonia. The frequency of fimbria-associated adhesins among respiratory pathogens has not been studied in detail. In this study isolates belonging to the family Enterobacteriaceae, prospectively obtained from intensive care unit patients who were suspected of having nosocomial pneumonia, were examined for fimbria-associated adhesins. Type 3, P, type 1, and other fimbrial phenotypes were identified by specific hemagglutination and electron microscopy. The Klebsiella type 3 fimbrial phenotype was further characterized by using a monoclonal antibody. Also, both type 3 and Escherichia coli P fimbrial genotypes were detected by using DNA colony blot assays. The frequencies of genera or species isolated were as follows: Enterobacter (38.6%), Klebsiella (26.8%), Serratia (17.7%), E. coli (13%), and Proteus (5.2%). Isolates of Klebsiella oxytoca, K. pneumoniae, and Enterobacter cloacae most commonly possessed the type 3 fimbrial phenotype and genotype. The phenotype and genotype for E. coli P fimbriae (46.2 and 50%, respectively), a known pathogenic determinant in the urinary tract, were detected more frequently than expected. In addition, a previously unspecified hemagglutinin that was specific for porcine erythrocytes was almost uniformly expressed among isolates of Enterobacter aerogenes. Finally, the expression of the type 1 fimbrial phenotype was widely detected among the isolates tested but notably absent among K. oxytoca and Proteus mirabilis isolates. The frequency of the various fimbrial types identified suggests a role for these bacterial organelles in adherence to respiratory epithelia.

Bacterial Adhesion↗

PubMed1990

Characterization of the type 1 fimbrial subunit gene (fimA) of Serratia marcescens.

The nucleotide sequence of a DNA fragment that contains the fimA gene, encoding the major fimbrial subunit, of Serratia marcescens IA506 and associated flanking sequences has been elucidated. In addition, the origin of transcription has been identified and is located 120 base pairs upstream of the fimA initiation codon. The predicted amino acid sequence of the FimA polypeptide exhibits some degree of sequence homology with the fimbrial subunits encoded by the fimA determinants of Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli and also to Smf2, the major structural component of mannose-resistant (MR) fimbriae of S. marcescens. The Serratia adhesin that facilitates haemagglutination mediated by type 1 fimbriae is less susceptible to inhibition by D-mannose than has been observed to be the case in other type 1 fimbrial adhesins. The molecule conferring this adherence specificity has been shown to be distinct from the fimA gene product and, therefore, is analogous to the fimbrial systems reported in other species.

Amino Acid Sequence↗

PubMed1990

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles.

Tissue-binding specificity of the type-3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild-type Klebsiella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type-3 fimbriae, as well as the purified type-3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type-3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type-3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type-3 fimbrial gene cluster in association with the pap-encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type-3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type-3 fimbriate strains of Yersinia and Salmonella, showing that the ability to use type V collagen as tissue target is widespread among enteric bacteria.

Acetates↗