Biomedical subjects
Publications and source records attributed to T F Smith.
In an effort to identify the structural elements essential to a given protein function a new pattern-directed inference system has been developed. It has been employed to identify a potential dinucleotide-binding domain within the human mitochondrial unidentified reading frame 6 product, thereby supporting an earlier study that this gene may encode a NADH dehydrogenase subunit.
Multiple serum specimens from 10 patients with known cytomegalovirus (CMV) infections and 498 sera consecutively submitted to the laboratory for the diagnosis of CMV infection were tested for anti-CMV IgM after treatment with goat anti-human IgG and QAE-Sephadex A50 column chromatography. Specimens from all 10 patients were positive for IgM after treatment with anti-IgG, but only 8 after the column procedure. Anti-CMV IgM was detected in 23 of 498 (4.6%) specimens pretreated with anti-IgG but in only 12 (2.4%) of these samples after the sera was passed through QAE-Sephadex A50 columns. Anti-CMV IgM was detected exclusively in 2 sera after QAE-Sephadex A50 column treatment (sensitivity, 83%) and in 13 specimens pretreated with anti-IgG sera (specificity, 97%). Serology (IgG and IgM) was compared with the shell vial cell culture assay and histology for providing the first evidence of CMV infection in 28 liver transplant patients. CMV infection was detected initially by the rapid shell vial assay (24) or histology (3) in 27 (96%) of these patients. Detection of anti-CMV IgM in these patients had little value for rapid diagnosis of these infections, and suggests that serology should be recommended mainly for the diagnosis of primary CMV infections in localities in which the rapid shell vial assay is not available.
Twelve liver and 5 kidney transplant recipients with severe cytomegalovirus infection were treated with Ganciclovir (7.5 mg/kg/day, intravenously). Ten were evaluable (compatible clinical picture, organ involvement shown histopathologically or by culture, viremia, and absence of concomitant infection). All 17 patients were studied for adverse drug side effects. A total of 9 evaluable patients survived the infection; 1 died during treatment due to infection or drug toxicity. A death 19 days after completion of treatment was due to unrelated causes. Patients became afebrile after 2-9 days (mean, 5.3 days) of treatment. Liver function improved, pulmonary infiltrates cleared, and hypoxemia reversed during therapy. Viremia ceased during therapy in 9 patients; asymptomatic viruria persisted or recurred in 6 of 7 patients studied. No relapses occurred during follow-up (7-17 months; mean, 13 months). Transient neutropenia and thrombocytopenia occurred in 3 and 1 patients, respectively. Ganciclovir appears promising for treatment of severe CMV infection in patients with kidney or liver transplants.
Specimens submitted for diagnosis of herpes simplex virus (HSV) infection were inoculated into shell vials and reacted with a commercial DNA probe kit (Pathogene; Enzo Biochem, Inc., New York, N.Y.) and an immunofluorescence assay at 16 h postinoculation. The results were compared with isolation of the virus in conventional tube cell cultures. Of 504 specimens, 105 (20.8%) were positive for HSV. Of the 105, 93 HSV-positive specimens (89%) were detected by all three assay systems. Maximum detection of HSV (100 of 105 [95%]) was obtained by probe or monoclonal antibody assay in shell vials, which had sensitivities of 98 and 97%, respectively, compared with viral recovery in conventional tube cell cultures (mean time for recognition of cytopathic effects, 2 days). Both shell vial assays were 99% specific. The DNA probe kit may be used as an alternative to a monoclonal antibody and fluorescence assay in shell vials as a diagnostic method for rapid laboratory detection of HSV infection.
Two density gradient separation techniques for separation of blood leukocytes were compared for the laboratory diagnosis of cytomegalovirus (CMV) viremia. Of 510 blood specimens processed by both methods, 76 (14.9%) yielded CMV. Of the 76 positive specimens, 66 (87%) and 65 (86%) were processed by the Ficoll-Paque/Macrodex (F-P/M; Macrodex is dextran 70 in normal saline; Pharmacia, Pisataway, N.J.) and Sepracell-MN methods, respectively. Of the 76 CMV-positive blood specimens, 72 (95%) were detected in shell vial cell cultures, whereas only 42 (55%) were detected in conventional tube cell cultures. The time for recognition of specific cytopathic effects due to CMV in tube cell cultures (8.0 versus 7.1 days), the number of fluorescent foci in each positive shell vial culture (19.3 versus 20.1), and the costs of the reagents ($3.50 versus $2.80) were similar and independent of the leukocyte separation method (F-P/M versus Sepracell-MN). Recovery of CMV from heparinized blood (F-P/M method) was similar to that from EDTA-anticoagulated blood (Sepracell-MN method). The Sepracell-MN method is a rapid and sensitive method for detection of CMV from blood specimens and is recommended as a replacement for the more tedious and time-consuming F-P/M procedure.
Endocervical samples were obtained from 435 females for Chlamydia detection by using both a Dacron swab and a cytobrush. Positive results were obtained from 35 swabs and 34 cytobrush specimens. All specimens positive with the cytobrush were detected also with swab samples. The cytobrush and Dacron swab appear to be comparable for the detection of endocervical infections with Chlamydia trachomatis.
Specimens submitted for the diagnosis of cytomegalovirus (CMV) infection were inoculated into three (blood) or two (urine, tissue, bronchoalveolar lavage [BAL]) shell vials seeded with MRC-5 cells for the diagnosis of CMV infection. We evaluated the detection of 993 specimens that were positive for CMV according to the number of shell vial cell cultures inoculated per specimen. For blood cultures, and considering one CMV-positive shell vial as 100%, inoculation of three shell vials versus one increased the detection rate of the virus by 51%. Inoculation of three shell vials compared with two yielded a 20% increase in the detection rate of CMV. For urine, tissue, and BAL specimens, inoculation of two shell vials compared with one resulted in increases of 7, 10, and 5%, respectively. For maximum detection of CMV in shell vial cell cultures, at least three vials should be inoculated with blood specimens, and two vials should be used for urine, tissue, and BAL samples.
Urine specimens submitted for the diagnosis of cytomegalovirus infection were inoculated into shell vials that had been pretreated with a combination of dimethyl sulfoxide (DMSO) and dexamethasone (DEX). The results were compared with those for inoculated shell vials which had received no drug treatment. Of 664 specimens, 100 (15%) were positive for cytomegalovirus. Of the 100 strains of cytomegalovirus, 88 (88%) were detected in both DMSO-DEX-treated and untreated shell vials. Of the remaining 12 positive specimens, 6 were detected with untreated shell vials exclusively and 6 were detected with DMSO-DEX-treated shell vials alone (not significant by the sign test). The median number of fluorescent foci was not significantly higher in DMSO-DEX-treated shell vials compared with that in untreated cultures (Wilcoxon signed-rank test; P = 0.1). DMSO-DEX-treated monolayers did not enhance the sensitivity detection of cytomegalovirus in shell vial cell cultures.
Regions containing similar elements of primary and predicted secondary structure were identified in simian virus 40 large T, adenovirus E1A, c-myc and v-myc proteins by a computer program with a set of highly specific, complex pattern descriptors. In all cases these regions were localized in domains of the respective proteins known to be required for transforming activity. We suggest that these apparently structurally similar regions may mediate a common biological function.
Block copolymers composed of polyoxyethylene and polyoxypropylene were found to increase the influx of Na+ and the efflux of K+ from human erythrocytes. They were, however, ineffective at promoting the transport of Ca2+. The size of the ion fluxes induced by the copolymers correlated with their efficacy in stimulating inflammation. These compounds were also found to induce conductance increases in planar lipid bilayers in a nonvoltage dependent and nonstepwise manner. In both experimental systems, ion transport was facilitated only under temperature and ionic-strength conditions in which the polymers form aggregates in aqueous solution. In neither system did the concentration dependence of transport activity exhibit a pronounced cooperativity. These observations are consistent with the view that aqueous monomers of these surface active agents partition into the membrane, where they facilitate the conductive movement of monovalent cations by means of a carrier type mechanism. As a novel class of ionophores, these substances are of practical interest because they can be water soluble and are potentially reversible.
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The aminoacyl-tRNA synthetases are united by a common function with little evidence of a common structural relationship. Outside of an 11 amino acid stretch called the "signature sequence", no global primary sequence similarity exists. The signature sequence matches 4-11 amino acids in several aminoacyl-tRNA synthetases. High-resolution X-ray data are available for two of these enzymes, revealing that their signature sequence regions are small segments of a common mononucleotide binding foldlike structure. A new methodology for the analysis of dissimilar primary sequences supports the expectation that all of the signature sequence regions form a common structure. In our analysis, two complex pattern descriptors were constructed to describe the synthetase mononucleotide binding fold. These were compared to primary sequences annotated with predicted secondary structures and hydropathy profiles. Regions in 8 out of 12 (67%) heterologous aminoacyl-tRNA synthetase groups (where each group is specific for the same amino acid) match the first descriptor, and 7 of these (58%) also match the second descriptor. In contrast, only 4 regions in a set of 54 control proteins (7.4%) match the first descriptor, and only 2 regions (3.7%) match both. Alignment of these 8 regions to the descriptor (1) positions all known signature sequence regions as the first loop of a mononucleotide binding foldlike structure, (2) extends the previous alignments by another 40-odd amino acids, and (3) identifies potential sites in 3 out of 6 heterologous aminoacyl-tRNA synthetases with no previous alignments. Potential sites are also proposed for two additional heterologous synthetases on the basis of matches to less specific descriptors.
The physicochemical properties of beta-turns suggest their biological importance prior to the formation of the genetic code. These properties include ones potentially affecting the preference for either L- or D-amino acids. The abundance of certain amino acids in beta-turns is correlated with their assignment to a small, well-defined part of the genetic code and with their role as metabolic precursors for other amino acids. It is proposed that in the prebiotic environment, beta-turns became objects of selection that influenced the evolution of the genetic code and biosynthetic pathways for amino acids.
A 1580-bp fragment of Staphylococcus aureus plasmid pUB110 encoding resistance to the DNA synthesis inhibitor bleomycin has been cloned and sequenced. A DNA sequence containing an open reading frame of 405 bp was subcloned into several expression vectors and bleomycin resistance was expressed at high level in Escherichia coli under the control of lambda PL promoter. On induction, a ca. 14,000-Da protein was detected by gel electrophoresis. The bleomycin resistance determinant of the gram-positive plasmid pUB110 was compared to that of the enterobacterial transposon Tn5; limited regions of close relatedness could be identified.
Use of Quik-Sep System II (QSII) ion-exchange chromatography increased the detection of CMV-directed immunoglobulin M (IgM) compared to the prototype Quik-Sep System (QSI) (27 of 87 [31%] IgM-positive versus 20 of 87 [24%]). However, an increase in the number of false-positive rheumatoid factor-containing sera was found (15/52 [29%] versus 8/52 [15%]). Total immunoglobulin G (IgG) and IgM concentrations were higher in the QSII eluates compared to those from QSI, although total IgM recovery did not exceed 36% in either system.
We prospectively evaluated the diagnostic role of bronchoalveolar lavage in the assessment of opportunistic pulmonary infections and the incidence of associated complications in 100 immunocompromised patients during a 9-month period. Bronchoalveolar lavage was useful in detecting the presence of Pneumocystis carinii, viruses, fungi, bacteria, and mycobacteria in the lower respiratory tract. P. carinii was diagnosed by bronchoalveolar lavage in 17 patients and by open-lung biopsy in 1. Other organisms detected by lavage, lung biopsy, or both included viruses (eight patients), fungi (four patients), bacteria (six patients), and mycobacteria (three patients). Of the 100 patients studied, 33 had infectious agents detected in the lung; in 6 of these patients, more than one organism was present. Bronchoalveolar lavage detected the infectious organisms in 30 of the patients, in many of whom an open-lung biopsy was likely avoided because of the lavage studies. Although no major complications of bronchoalveolar lavage were noted in this critically ill population, five patients did require short-term mechanical ventilation after bronchoscopy. When correctly used, bronchoalveolar lavage is a safe and useful procedure for the assessment of immunocompromised subjects with suspected opportunistic pulmonary infections.
The sensitivity of an indirect fluorescent antibody (IFA) test (screening test) for the detection of antibodies to cytomegalovirus (CMV) was examined by using 128 serum specimens and quaternary aminoethyl (QAE)-Sephadex A50 column chromatography to separate IgM from IgG class antibodies. Of these 128 rheumatoid factor-negative serum samples, only 54 (42%) continued to produce fluorescence after the IgG was removed by the Sephadex column (yielding a theoretical 1:10 dilution) and the samples were retested for IgM (confirming test). Of the 54 specimens that were positive on both the screening and confirming IgM IFA assays, 53 (98%) had titers of 1:80 or more. Of the 74 sera that were negative on the confirming test, only 10 (14%) had titers of 1:80 on the initial screening test, and none was greater than 1:80. Immunoglobulin measurements of eight serum samples before and after column treatment indicated excellent IgG removal but poor IgM recovery (a mean loss of 85% of the total IgM). Comparison of the time needed to detect IgM in CMV culture-positive patients demonstrated an increase from 17.1 to 28.3 days (serum tested by IFA before and after column separation, respectively). Therefore, confirmation of anti-CMV IgM in serum specimens by QAE-Sephadex A50 column chromatography seems to be of low sensitivity.
The location and sequence composition of a consensus element of the nucleotide binding site in both simian virus 40 (SV40) and polyomavirus (PyV) large tumor antigens (T antigens) can be predicted with the assistance of a computer-based pattern-matching system, ARIADNE. The latter was used to optimally align elements of T antigen primary sequence and predicted secondary structure with a "descriptor" for a mononucleotide binding fold. Additional consensus elements of the nucleotide binding site in these two proteins were derived from comparisons of T antigen primary and predicted secondary structures with x-ray structures of the nucleotide binding sites in four otherwise unrelated proteins. Each of these elements was predicted to be encompassed within a 110-residue segment that is highly conserved between the two T antigens residues 418-528 in SV40 T antigen and residues 565-675 in PyV). Results of biochemical and immunologic experiments on the nucleotide binding behavior of these proteins were found to be consistent with these predictions. Taken together, the latter have resulted in a topological model of the ATP binding site in these two oncogene products.