Biomedical subjects
Publications and source records attributed to M Cantrell.
A decade of organizing, implementing, and evaluating interdisciplinary care teams at St Luke's Regional Medical Center has reinforced the value of developing and maintaining clinical and nonclinical competencies. The value of consensus in task delegation cannot be overemphasized. Unit directors have expanded their scope of responsibility and must remain vigilant to avoid fragmentation of care in an interdisciplinary team delivery system.
Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.
The clinical skills of sophomore medical students at the University of Arkansas are being assessed through the use of the Objective Structured Clinical Examination (OSCE). This exam was developed in order to better standardize the evaluation of practical clinical skills. The exam uses standardized patients, who are lay people trained to accurately and consistently portray a patient encounter. Faculty members at UAMS authored clinical cases for 20 patient encounters that test history taking, physical examination and communication skills. Each student interacts with the patient while being assessed in a standardized way, and then is given educational feedback by a faculty member. Students who do not pass the exam, undergo a remediation program prior to entering the junior year.
In response to the current emphasis on health maintenance and disease prevention, the authors developed a comprehensive education program in which sophomore medical students interview a standardized patient about breast problems and risk factors, receive one-on-one instruction from the standardized patient during the clinical breast examination, and practice recommendations for screening and instruction in breast self-examination. In this pilot study sophomore students who underwent the comprehensive education program were compared with students who received the traditional, didactic instruction and practiced on plastic breast models. The students who received the didactic instruction had mean scores on a multiple-choice knowledge-base pretest and posttest of 54.6% and 76.8%, respectively. The students who participated in the comprehensive education program had mean pretest and posttest scores of 51.2% and 78.5%, respectively. All students participated in a practical test of the clinical breast examination during an objective structured clinical examination. The students who had received the didactic instruction scored 69.9% (mean), compared with 84.1% for the students who had had the comprehensive education program. The comprehensive breast education program teaches medical students about risk factors, screening recommendations, and clinical breast examination more effectively than do traditional didactic methods.
The genes encoding phytase (EC 3.1.3.8) and pH 2.5-optimum acid phosphatase (EC 3.1.3.2) have been cloned and sequenced from Aspergillus niger var. awamori. The translated nucleotide sequences yielded polypeptides of 467 and 479 amino acids (aa) for phytase and acid phosphatase, respectively. The genes were isolated using oligodeoxyribonucleotide probes based on the aa sequences of the purified proteins. Recombinant A. niger var. awamori strains carrying additional copies of the gene sequences demonstrated elevated enzyme activities.
A panel of 12 monoclonal antibodies were produced by immunization of Balb/c mice with small bowel epithelial cells obtained from a patient with active well-documented Crohn's disease. The clones were derived by screening with immunofluorescence microscopy; those with staining patterns suggestive of mucin directed epitopes were chosen for study. Several distinct patterns of staining reactivity were noted, including reagents with homogeneous, luminal, heterogeneous and peripheral goblet cell activity. In addition, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis revealed reactivity to high molecular weight mucin. The reactive antigen was resistant to proteinase digestion. No endoneuraminidase activity was detected; however, one neuraminidase sensitive sialic acid epitope was demonstrated. Confirmation of glycoprotein epitopes was accomplished by testing purified mucins from several areas of the gastrointestinal tract by ELISA. Finally, individual small bowel goblet cell heterogeneity was demonstrated by immunofluorescence, Western blotting, and antibody affinity chromatography. These data demonstrate both by morphology and specific binding of antibody affinity chromatography a significant degree of small bowel goblet cell mucin heterogeneity.
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Previous observations have purported to demonstrate circulating antibodies which bind to colon epithelial cells. However, the significance and reproducibility of such observations has been difficult and the data often phenomenological. To further our understanding of such autoreactivity, we studied sera and purified serum immunoglobulins from patients with ulcerative colitis, Crohn's colitis and other inflammatory diseases, as well as normal volunteers using as a target, well-defined epithelial cell preparations from normal and diseased colon and small bowel including crude suspensions of homogenized cells, purified and characterized brush border membranes, basolateral membranes and a DEAE cellulose column purified protein fraction. Homogenates of normal liver, lung, kidney, thymus, pancreas, stomach and small and large intestine, obtained at surgery, were also included. The purified preparations were characterized by enzyme activity and were electrophoresed on SDS-polyacrylamide gels for immunoblotting. Additional studies were carried out comparing these findings with those of a previously published and described 'positive' colon target preparation and polyclonal antibody. There was no convincing demonstration of circulating autoantibodies in patients with ulcerative colitis. Our data, using well-defined and characterized tissue preparations, raises doubts regarding the presumptive demonstration of autoantibodies in ulcerative colitis.
Three human small bowel and colon mucosal specific monoclonal antibodies with distinct morphologic and electrophoretic characteristics were generated by fusion of immunized Balb/c spleen cells and murine plasmacytoma cells. Morphologic specificity by indirect immunofluorescence (IIF) revealed three antibody binding patterns corresponding to villus surface (TP-NG-43), goblet cell apical granules (TP-NG-2), and a combined surface/goblet cell apical granule antibody (TP-NG-20). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) produced three distinct electrophoretic migration patterns. These antibodies reacted with very high molecular weight determinants: TP-NG-2, one band greater than 400 kD; TP-NG-20, two bands corresponding to 370-400 kD; and TP-NG-43, two bands in the 350-400-kD range with smaller bands in the 50-94-kD range. Cross-reactivity with various other human organ systems was evaluated by indirect immunofluorescence and SDS-PAGE electrophoresis with Western blotting. By IIF, all three monoclonal antibodies reacted very strongly with components of gastric mucosa. Weak cross-reactivity was seen with colon, rectum and mucin-producing adenocarcinoma of the colon. No cross-reactivity was observed by IIF with other mucin-containing and non-mucin-containing tissues. However, cross-reactivity with gastric mucin was not detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antibody reactivity with mucin was confirmed by purifying various regional gastrointestinal mucins and by subsequent testing by ELISA. Monoclonal antibody affinity columns were prepared and evaluated. The utility of these methods will allow for further definition of important goblet cell mucin glycoprotein characteristics and isolation of mucin subpopulations.
Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.
Twenty elderly nursing home patients with long-term indwelling bladder catheters were studied to evaluate the validity of the microbiology of urine samples obtained from catheters that had not been changed for at least 30 days. Paired urine samples from "old" catheters and newly inserted catheters were compared for quantitative and qualitative microbiology. Urine microbiology for old catheters was highly sensitive but had poor specificity.
Infective endocarditis has become a disease affecting primarily elderly persons. The etiology of infective endocarditis in the elderly is predominantly streptococci and staphylococci. The clinical features of this infection in the aged may be atypical or nonspecific, which often leads to delays in diagnosis. Mortality is extremely high for elderly patients with infective endocarditis.
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In a previous publication1 we reported that the tyrosine selective reagent, tetraitromethane, causes complete inactivation of E. coli 30S ribosomes for poly U directed non-enzymatic phe-tRNA binding. This inactivation was demonstrated to be due to the chemical modification of the protein moiety of the ribosome. We have no identified the proteins of the 30S particle inactivated by this modification. Using a method of ribosome reconstruction we have found that unmodified proteins S1, S11, and S21 are essential for the restoration of the phe-tRNA binding activity of tetranitromethane inactivated ribosomes. We propose that these three proteins are intimately involved in the 30S ribosome binding site for tRNA.
30S ribosomal protein S4 contains a single cysteine residue at position 31. We have selectively cleaved the peptide bond adjacent to this residue using the reagent 2-nitro-5-thiocyanobenzoic acid. The two resultant fragments were purified. The smaller S4-fragment (1-30) was found to be incapable of interacting with 16S RNA directly. This fragment also is not incorporated into a particle reconstituted from 16S RNA and 20 purified proteins with S4 missing. In contrast, the large S4-fragment (31-203) appears to be fully functional in ribosome assembly. Replacement of S4 with this fragment in the reconstitution reaction leads to a complete 30S ribosome containing all 30S proteins. This particle has a full capacity to bind poly U but has lost all activity for poly U directed phe-tRNA binding. We therefore propose that the N-terminus of protein S4 is not critical for ribosome assembly but is essential for tRNA binding.
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