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T F Smith

Publications and source records attributed to T F Smith.

At least 37 records · Page 2Linked to original sources
PubMed2000

Thirty-plus functional families from a single motif.

It is now possible to identify over 30 functional subfamilies among the WD-repeat-containing proteins found in the completed genomes. The majority of these subfamilies have at least one member for which experimental data allow assignment to a cellular pathway or process. Half of the 63 WD-repeat-containing proteins in Saccharomyces cerevisiae, half of the 70 in Caenorhabditis elegans, and a third of the 100 plus predicted in Drosophila can be assigned to 23 of these functional subfamilies. Perhaps indicative of the future, 33 WD-repeat-containing proteins from the partial genome of Arabidopsis thaliana can now be assigned to 18 of these subfamilies. These assignments have been made possible by combining traditional sequence similarity with an implied common beta propeller structural context to obtain measures of protein-protein surface similarity. The beta propeller structural context is represented in the form of a Hidden Markov Model. The procedure is completely automated.

Algorithms↗
PubMed2000

Analytical performance and clinical utility of a nucleic acid sequence-based amplification assay for detection of cytomegalovirus infection.

A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection.

Antigens, Viral↗
PubMed2000

Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation.

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.

Cytomegalovirus↗
PubMed2000

Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR.

Herpes simplex virus (HSV) causes several clinical manifestations in both normal and immunocompromised hosts; this agent is the most frequently detected virus in diagnostic laboratories. Recovery of the virus in cell culture is considered the "gold standard" for detection of this virus from sources other than cerebrospinal fluid. LightCycler is a newly developed, commercially available system designed to rapidly perform PCR, with real-time detection of PCR products by a fluorescence resonance energy transfer assay. We compared the detection of HSV for 200 specimens (number of genital specimens, 160; number of dermal specimens, 38; number of ocular specimens, 2) by shell vial cell cultures (MRC-5) and by LightCycler PCR. Of a total of 88 (44%) HSV strains detected, 69 (78%) were detected by both shell vial cell cultures and LightCycler PCR (DNA polymerase target). A total of 19 (22%) specimens were detected exclusively by LightCycler PCR. No specimens were positive by the shell vial assay only. All 19 discrepant samples had HSV DNA detected by an independent PCR directed to the thymidine kinase gene of the virus. The melting curve analysis feature of the LightCycler instrument identified identical genotype results for HSV type 1 (HSV-1) and HSV-2 from 84 of 88 (96%) positive samples. Specimens can be extracted, target HSV DNA can be amplified, and HSV PCR products can be identified by genotype within 2 h after receipt of specimen into the laboratory. The increased level of accurate identification (all 88 positive samples) compared with that of shell vial cell culture (69 of 88 samples identified as positive) and the agreement of LightCycler PCR results with all shell vial positive results indicate the potential for routine implementation of this technology for laboratory diagnosis of HSV infections.

DNA, Viral↗
PubMed2000

Evaluation of LightCycler PCR for implementation of laboratory diagnosis of herpes simplex virus infections.

Five hundred specimens (288 genital, 192 dermal, and 20 ocular) were extracted by technologists, and the DNA was assayed by LightCycler PCR (DNA polymerase and thymidine kinase [TK] gene targets) and by conventional tube and shell vial cell culture. One hundred fifty-eight confirmed (by cell culture and TK target PCR) positive and LightCycler-positive specimens were detected during the first 30 PCR cycles. LightCycler PCR-positive results for cycles 31 to 45 (39 of 67 [58.2%]) required confirmation by another PCR target (TK). LightCycler PCR is more sensitive (n = 197; 23.1%) than cell cultures (n = 150) for the routine laboratory detection of herpes simplex virus infections.

DNA-Directed DNA Polymerase↗
PubMed2000

Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR.

Varicella-zoster virus (VZV) causes vesicular dermal lesions which are clinically evident as varicella (primary infection) or zoster (reactivated) diseases. The LightCycler system (Roche Molecular Biochemicals) is a newly developed commercially available system designed to rapidly perform PCR with real-time detection of PCR products using a fluorescence resonance energy transfer. We compared the detection of VZV from dermal specimens by shell vial cell culture (MRC-5) and by LightCycler PCR. Of 253 specimens, VZV was detected in 23 (9.1%) by shell vial cell cultures and 44 (17.4%) by LightCycler PCR directed to a nucleic acid target sequence in gene 28. Twenty-one of 44 (47.7%) specimens were exclusively positive by LightCycler PCR; the shell vial cell culture assay was never positive when DNA amplification was negative (specificity, 100%). VZV DNA was detected in 39 of 44 (88.6%) specimens positive during cycles 10 through 30 of the LightCycler PCR. These VZV DNA-positive specimens (cycles 10 to 30) and 5 of 11 other PCR positive specimens (cycles 31 to 36) were confirmed by another LightCycler PCR directed to another (gene 29) target of the viral genome. For routine laboratory practice, all specimens yielding amplified DNA to the VZV gene 28 target can be considered positive results. The increased sensitivity (91%) of the LightCycler PCR for detection of VZV, rapid turnaround time for reporting results, virtual elimination of amplicon carryover contamination, and equivalent costs compared to shell vial cell culture for detection of VZV indicate the need for implementation of this technology for routine laboratory diagnosis of this viral infection.

Chickenpox↗
PubMed2000

Eliminating superfluous neighbor pairs while threading fold models.

In this paper we address the problem of identifying which of various possible spatial residue-residue neighbor pairs are plausible physical contacts without reference to the native structure side chain geometry. We propose an algorithm that eliminates most of the implausible physical contacts from the fold models. This algorithm exploits the correlations between the amino acid side chain rotamers and the direction of the physical contacts between the amino acid side chains. We use this algorithm to "filter" the score of the sequence-to-structure alignment. Filtering is dynamic, in the sense that the set of neighbor pairs contributing to the alignment score varies during threading. Whether or not a neighbor pair contributes to the score depends on the threaded amino acids. This score filtering improves the accuracy of the predicted sequence-to-structure alignment.

Algorithms↗
PubMed1999

In silico detection of control signals: mRNA 3'-end-processing sequences in diverse species.

We have investigated mRNA 3'-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3'-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3'-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3'-end-processing signals will require more than consensus sequence identification.

Animals↗
PubMed1999

Filtered neighbors threading.

We present a knowledge-based threading scoring function that exploits the information about protein structure contained in residue packing/neighbor preferences. The proposed algorithm eliminates the stereochemically improbable physical contacts for each possible sequence-to-structure alignment. We use this algorithm to "filter" the score of the sequence-to-structure alignment. Filtering is dynamic, in the sense that the set of neighbor pairs contributing to the alignment score varies during threading. Whether or not a neighbor pair contributes to the score depends on the threaded amino acids. We use a detailed structure description that encodes amino acid side-chain rotamer and physical contact preferences but does not imprint the fold model with the native sequence or native physical contacts. We discretize this description to collect accurate statistics for the scoring function generation. We use the original detailed description for the neighbor filtering. On average, the filtered neighbors threading (FNT) method predicts the sequence-to-structure alignment twice as accurately as does the "standard" unfiltered neighbors threading. For the set of threadings tested by the PHDthreader method, the FNT gives predictions with a sequence-to-structure alignment accuracy of 46.9%, which amounts to a 74% improvement in alignment sensitivity compared with PHDthreader predictions. These results show that reduction of noise from the observed neighbor pair preferences by filtering leads to noticeable improvements in the predicted sequence-to-structure alignments.

Algorithms↗
PubMed1999

Relationship of HHV8 replication and Kaposi's sarcoma after solid organ transplantation.

HHV8 DNA sequences have recently been isolated from all types of Kaposi's sarcomas, and its association in the etiopathogenesis of this tumor has been established. However, little is known about the regulation of HHV8 replication in immunocompromised patients seropositive for this virus, and its impact on the development of Kaposi's sarcoma (KS). Through the study of a heart transplant patient who developed KS and in whom peripheral blood lymphocytes (PBLs) had been prospectively collected before and after transplantation, we have investigated the pathogenesis of HHV8. Our results indicate that (i) HHV8 can reactivate soon after transplantation; (ii) viral replication, as determined by quantification of HHV8 DNA load of PBLs, increases significantly after transplantation; and (iii) increased HHV8 DNA levels in PBLs are associated with the development of KS.

DNA, Viral↗
PubMed1999

Novel mutation in the CMV UL97 gene associated with resistance to ganciclovir therapy.

Cytomegalovirus (CMV) strains resistant to ganciclovir have been associated with specific mutations in the UL97 and UL54 genes. The UL97 gene of a CMV strain isolated from a renal transplant recipient before and after 438 days of ganciclovir treatment was amplified by polymerase chain reaction and sequenced. A novel mutation resulting in deletion of codons 595 to 603 was identified in the viral DNA from specimens obtained after, but not before, prolonged ganciclovir therapy. Clinical and virological resolution of CMV disease occurred after switching to foscarnet therapy. Although many ganciclovir resistance mutations have been mapped to the UL97 codon range 591-607, this one is unusual in that it involves deletion of half these codons. Because UL97 seems to be necessary for effective CMV replication, this deletion suggests that much of codons 591-607 can be removed without destroying the biological function of UL97, and that this codon range can be altered in various ways to affect ganciclovir susceptibility. Rapid, flexible genotypic assays directed at this part of UL97 may facilitate the early recognition of ganciclovir resistance.

Adult↗
PubMed1999

Human herpesvirus 6 seronegativity before transplantation predicts the occurrence of fungal infection in liver transplant recipients.

BACKGROUND: Invasive fungal infection has a major impact on the morbidity and mortality of liver transplant recipients. Human herpesvirus (HHV)-6 infection after transplantation is associated with an immunosuppressive state and the development of cytomegalovirus disease. Because cytomegalovirus infection is a risk factor for invasive fungal infection after transplantation, we have examined whether HHV-6 and fungal infection are associated after transplantation. METHODS: Pretransplantation sera from 247 consecutive liver transplant recipients were analyzed for IgG to HHV-6. Thirty-three (13%) HHV-6-seronegative recipients were identified. Six of 33 (18%) seronegative recipients experienced fungal infection as compared with 15 of 214 (7%) seropositive recipients (P=0.034). RESULTS: In a univariate analysis of risk factors for fungal infection, pretransplantation seronegativity to HHV-6 (P=0.034), intraoperative cryoprecipitate requirements greater than the 75th percentile (P=0.035), reoperation (P=0.005), biliary stricturing postoperatively (P=0.046), and gastrointestinal or vascular complications postoperatively (P=0.030) were identified as significant risk factors. Moreover, in pairwise multivariate analysis, pretransplantation HHV-6 seronegativity remained a significant variable even in the presence of each of the other variables. CONCLUSIONS: These results suggest that HHV-6 seronegativity before transplantation is a valuable clinical marker that identifies patients at risk for developing fungal infection after transplantation.

Disease-Free Survival↗
PubMed1999

Genomic detection of new yeast pre-mRNA 3'-end-processing signals.

To investigate Saccharomyces cerevisiae 3'-end-processing signals, a set of 1352 unique pre-mRNA 3'-end-processing sites, corresponding to 861 different genes, was identified by alignment of expressed sequence tag sequences with the complete yeast genome. Nucleotide word frequencies in the vicinity of the cleavage sites were analyzed to reveal the signal element features. In addition to previously recognized processing signals, two previously uncharacterized components of the 3'-end-processing signal sequence were discovered, specifically a predominance of U-rich sequences located on either side of the cleavage site. One of these, the downstream U-rich signal, provides a further link between the 3'-end-processing mechanisms of yeast and higher eukaryotes. Analysis of the complete set of 3'-end-processing sites by means of a discrimination function supports a 'contextual' model in which the sum total effectiveness of the signals in all four elements determines whether or not processing occurs.

Expressed Sequence Tags↗
PubMed1999

The role of parvovirus B19 in the pathogenesis of giant cell arteritis: a preliminary evaluation.

OBJECTIVE: To determine whether parvovirus B19 DNA is more likely to be present in the temporal arteries of patients with giant cell arteritis (GCA) than in the temporal arteries of control subjects. METHODS: We prospectively examined temporal artery biopsy (TAB) tissue from 50 consecutive patients presenting for TAB for the presence of B19 DNA using the polymerase chain reaction (PCR). Clinical and demographic information was obtained from the patients' medical records. A separate PCR analysis of 30 original tissue specimens was conducted at the Centers for Disease Control and Prevention (CDC) using primers directed toward another target sequence in the nonstructural coding area of B19. RESULTS: The 50 patients had an average age of 70.8 years; 27 (54%) were female. Amplicons for human beta-globulin, but not for cytomegalovirus, were produced for all tissue samples. The PCR results for B19 agreed in 29 of 30 samples tested by our institution and by the CDC (97% agreement; kappa = 0.9). A comparison of the B19 DNA analysis and the results of TAB indicated a statistically significant association between histologic evidence of GCA and the presence of B19 DNA in TAB tissue (chi2 = 10.38, P = 0.0013). CONCLUSION: These findings suggest that B19 may play a role in the pathogenesis of GCA.

Aged↗
PubMed1999

Laboratory diagnosis of Pneumocystis carinii infections by PCR directed to genes encoding for mitochondrial 5S and 28S ribosomal RNA.

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis carinii in clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage 1(BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.

Aged↗
PubMed1999

A colorimetric microtiter plate PCR system detects respiratory syncytial virus in nasal aspirates and discriminates subtypes A and B.

We developed a colorimetric microtiter plate (MTP) PCR system for specific detection of the respiratory syncytial virus (RSV) nucleocapsid gene and differentiation of viral subtypes A and B. Of 47 pediatric nasal aspirate specimens, the sensitivity and specificity were 94.4% (17 of 18) and 100% (15 of 15), respectively, when compared with RSV cell culture isolation in HEp-2 cells. An additional 14 specimens positive for adenoviruses, rhinoviruses, influenza, or parainfluenza viruses did not give positive reactions. PCR testing detected a mean of 0.15 (0.01 to 7.00) plaque-forming units of RSV virions. Inhibition of PCR amplification was detected in 33.3% (6/18) of undiluted specimens and could be avoided by a dilution (1:10) of extracted RNA without decreasing test sensitivity. RSV subtype, as determined by allele-specific probes, was identical to that determined by an immunofluorescence assay. These results indicate that the MTP PCR system provides a sensitive and specific test for clinical laboratory diagnosis and simultaneous subgroup classification of RSV infection.

Cell Culture Techniques↗
PubMed1999

The WD repeat: a common architecture for diverse functions.

Our knowledge of the large family of proteins that contain the WD repeat continues to accumulate. The WD-repeat proteins are found in all eukaryotes and are implicated in a wide variety of crucial functions. The solution of the three-dimensional structure of one WD-repeat protein and the assumption that the structure will be common to all members of this family has allowed subfamilies of WD-repeat proteins to be defined on the basis of probable surface similarity. Proteins that have very similar surfaces are likely to have common binding partners and similar functions.

Animals↗