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T F Smith

Publications and source records attributed to T F Smith.

At least 55 records · Page 3Linked to original sources
PubMed1999

Effective use of polymerase chain reaction for diagnosis of central nervous system infections.

Polymerase chain reaction (PCR)-based testing of cerebrospinal fluid (CSF) specimens has become standard for confirmatory diagnosis of central nervous system (CNS) infections; however, these tests increase health care costs. We reviewed 3-year data from 974 consecutive CSF specimens submitted for detection of seven pathogens by PCR. In 1997, 237 of 367 specimens (64.6%) were submitted for multiple tests, compared with 203 of 522 (38.9%) in 1996 and 18 of 85 (21.2%) in 1995. In each year the arrival of new house officers coincided with a peak in multiple testing. Among 732 specimens submitted for herpesvirus detection, results were positive for 24 (4.6%) of 523 specimens with increased leukocyte counts or protein levels. None of 209 specimens with normal leukocyte and protein levels were positive for herpesviruses. None of 471 CSF specimens submitted for Borrelia burgdorferi detection were PCR-positive. Use of protein and leukocytes to screen CSF specimens before employing PCR for herpesvirus detection would save almost one-third of costs without reducing sensitivity.

Central Nervous System Infections↗
PubMed1999

Positional statistical significance in sequence alignment.

Beginning with the concept of near-optimal sequence alignments, we can assign a probability that each element in one sequence is paired in an alignment with each element in another sequence. This involves a sum over the set of all possible pairwise alignments. The method employs a designed hidden Markov model (HMM) and the rigorous forward and forward-backward algorithms of Rabiner. The approach can use any standard sequence-element-to-element probabilistic similarity measures and affine gap penalty functions. This allows the positional alignment statistical significance to be obtained as a function of such variables. A measure of the probabilistic relationship between any single sequence and a set of sequences can be directly obtained. In addition, the employed HMM with the Viterbi algorithm provides a simple link to the standard dynamic programming optimal alignment algorithms.

Adenylate Kinase↗
PubMed1999

Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir.

Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) have been associated with resistance to acyclovir (ACY) and possible recognition of neurotropic strains. We sequenced a 335-bp segment of the TK gene to determine the frequency of mutations in HSV strains recovered from dermal, genital, and cerebrospinal fluid (CSF) specimens (n = 200; 102 HSV type 1 [HSV-1] 98 HSV-2 strains). Four polymorphic sites were detected in HSV-1 strains; C513T, A528G, C575T, and C672T. Among the polymorphisms, only C575T resulted in a change of amino acid sequence (residue 192, Ala-->Val). For HSV-2 strains, only one polymorphism (G420T) which resulted in an amino acid substitution (residue 139, Leu-->Phe) was detected. Phenotypic determination of resistance to ACY by a plaque reduction assay of 48 HSV isolates was not correlated with the sequence results of 11 strains in that 7 of these with genotypic polymorphisms were susceptible to the drug in vitro. In addition, of 32 ACY-resistant HSV strains, 28 (87.5%) had no polymorphisms detected in the 335-bp amplicon of the TK gene. There was no statistical difference in the frequency of polymorphisms according to the source of the specimens. We conclude that the detection of nucleic acid polymorphisms in a previously implicated 335-bp segment of the TK gene cannot be interpreted as indicative of either ACY resistance or neurotropism of HSV strains from dermal, genital, and CSF sources.

Acyclovir↗
PubMed1999

Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and </=0.900) were retested; if the second analysis produced an index of >0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests which require subjective interpretation, like the FTA-ABS test, may be biased by RPR test results.

Adult↗
PubMed1999

Evolutionary constraint networks in ligand-binding domains: an information-theoretic approach.

Ligand-binding sites in homologous protein domains can diverge greatly during evolution. This poses a particularly interesting problem in those cases where the ligand-binding site is situated in, or close to, the domain core, or where ligand-docking induces dramatic conformational changes. These features are present in many receptors and enzymes; the hormone-binding domain of the nuclear receptors for steroids and retinoids, for example, exhibits both characteristics. It is therefore of great interest to determine how binding sites for diverse ligands evolve in core regions of structurally dynamic domains. Are evolutionary changes locally restricted to the ligand-binding site, or are they distributed throughout the domain? We describe here an information-theoretic approach for the study of covariation between ligand-contacting residues and compensatory mutations that preserve the structural integrity and the conformational dynamics of ligand-binding domains. We apply this method to the analysis of the nuclear receptor ligand-binding domain and show that the ligand-contacting residues in the hormone-binding pocket are evolutionarily linked to an extensive network of covarying positions.

Amino Acid Sequence↗
PubMed1999

Human herpesvirus 6.

Human herpesvirus (HHV) 6 is a beta-herpes, DNA virus. This virus shows closest homology with cytomegalovirus and HHV-7. Infection usually occurs in infants 6 to 24 months of age, and primary infection may result in roseola. HHV-6 infection in infants is the commonest cause of fever-induced seizures. Infection in adults is seen primarily in immunocompromised hosts with solid organ transplants or in those with human immunodeficiency virus infection. The virus is capable of pronounced interaction in vitro with cytomegalovirus and human immunodeficiency virus and induces immunosuppression and apoptosis. The importance of these interactions in vivo necessitates further investigation. HHV-6 infection may contribute to the pathogenesis of multiple sclerosis. HHV-6 may be diagnosed by viral culture, serology, or polymerase chain reaction.

Herpesviridae Infections↗
PubMed1999

Talonavicular arthrodesis for correction of posterior tibial tendon dysfunction.

The awareness of PTTD has increased because of the efforts of McGlamry and Mueller. The treatment for PTTD depends on the patient's age and weight, systemic factors, length of time of the disease course, and the extent of foot collapse. The period of time from injury to diagnosis often is delayed because of the gradual progression of the condition. The patient that presents with an acute injury often responds well to a soft-tissue procedure. The delay in treatment usually necessitates the performance of an osseous procedure to correct the deformity and align the foot. The talonavicular arthrodesis is indicated in the flexible flatfoot deformity when degenerative changes of the subtalar joint are not present. The talonavicular arthrodesis is effective for correcting the flexible flatfoot deformity because it reduces the forefoot abduction, increases the height of the arch, stabilizes the medial column, and prevents excessive subtalar joint pronation. The primary complications associated with the talonavicular arthrodesis are nonunions and development of arthritis in adjacent joints. The incidence of nonunion can be directly attributed to poor surgical technique and early weight bearing during the postoperative period. The degenerative changes that occur in adjacent joints are often present preoperatively because of the long-standing valgus deformity. The procedure effectively maintains the correction of the flatfoot over a long period of time, and allows the patient to return to a pain-free lifestyle. The talonavicular arthrodesis is the procedure of choice in the flexible flatfoot deformity because the procedure corrects the malalignment of the subtalar and midtarsal joints and prevents excessive subtalar joint pronation.

Adult↗
PubMed1998

Sites important for PLCbeta2 activation by the G protein betagamma subunit map to the sides of the beta propeller structure.

The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.

Adenylyl Cyclases↗
PubMed1998

Sites for Galpha binding on the G protein beta subunit overlap with sites for regulation of phospholipase Cbeta and adenylyl cyclase.

Heterotrimeric G proteins, composed of alpha and betagamma subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free betagamma activates downstream targets, but its action is terminated by association with GDP-liganded alpha subunits. Because alpha can inhibit activation of many effectors by betagamma, it is likely that the alpha subunit binding surfaces on betagamma overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on beta shown to contact alpha in the recently published crystal structures of the alphabetagamma heterotrimer (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The alpha subunit binds to the flat, top surface of the toroidal beta subunit and also extends a helix along the side of the beta subunit at blade 1. We mutated four residues on the top surface of beta (Hbeta1[L117A], Hbeta1[D228R], Hbeta1[D246S], and Hbeta1[W332A]) and two residues on the side of beta that contacts alpha (Hbeta1[N88A/K89A]). Each of the mutant proteins was able to form beta gamma dimers, but they differed in their ability to bind alpha and to activate phospholipase C beta2 (PLCbeta2), PLCbeta3, and adenylyl cyclase II. Mutation of residues along the side of the torus at blade 1 diminish affinity for alpha but do not prevent activation of any of the effectors. Mutations on the alpha binding surface differentially affected PLCbeta2, PLCbeta3, and adenylyl cyclase II. Residues that affect PLCbeta and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the alpha subunit, make many contacts on the top surface. None of the mutations affected the ability of betagamma to inhibit adenylyl cyclase I. We conclude that alpha, PLCbeta2, PLCbeta3, and adenylyl cyclase II share an interaction on the top surface of beta. The importance of individual residues is different for alpha binding and for effector activation and differs even between closely related isoforms of the same effector.

Adenylyl Cyclases↗
PubMed1998

Clinical significance of viral load in the diagnosis of cytomegalovirus disease after liver transplantation.

In a cohort of 43 liver transplant recipients who did not receive antiviral prophylaxis, qualitative and quantitative polymerase chain reactions (PCRs) from peripheral blood were prospectively compared to determine their value in the diagnosis of established cytomegalovirus (CMV) disease and for the early detection of CMV replication as a marker for preemptive antiviral therapy. Using a cutoff of 7000 copies of CMV DNA per sample, the specificity and positive predictive values of qualitative PCR for the diagnosis of established CMV disease increased from 33% to 89% and from 54% to 82%, respectively, without reducing the 100% sensitivity and negative predictive value. By contrast, quantification of viral load provided no additional advantage to qualitative PCR for the early diagnosis of CMV infection before development of disease.

Blood↗
PubMed1998

Folding a WD repeat propeller. Role of highly conserved aspartic acid residues in the G protein beta subunit and Sec13.

The beta subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of an amino-terminal alpha-helical segment followed by seven repeating units called WD (Trp-Asp) repeats that occur in about 140 different proteins. The seven WD repeats in Gbeta, the only WD repeat protein whose crystal structure is known, form seven antiparallel beta sheets making up the blades of a toroidal propeller structure (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369-374). It is likely that all proteins with WD repeats form a propeller structure. Alignment of the sequence of 918 unique WD repeats reveals that 85% of the repeats have an aspartic acid (D) residue (not the D of WD) in the turn connecting beta strands b and c of each putative propeller blade. We mutated each of these conserved Asp residues to Gly individually and in pairs in Gbeta and in Sec13, a yeast WD repeat protein involved in vesicular traffic, and then analyzed the ability of the mutant proteins to fold in vitro and in COS-7 cells. In vitro, most single mutant Gbeta subunits fold into Gbetagamma dimers more slowly than wild type to a degree that varies with the blade. In contrast, all single mutants form normal amounts of Gbetagamma in COS-7 cells, although some dimers show subtle local distortions of structure. Most double mutants assemble poorly in both systems. We conclude that the conserved Asp residues are not equivalent and not all are essential for the folding of the propeller structure. Some may affect the folding pathway or the affinity for chaperonins. Mutations of the conserved Asp in Sec13 affect folding equally in vitro and in COS-7 cells. The repeats that most affected folding were not at the same position in Sec13 and Gbeta. Our finding, both in Gbeta and in Sec13, that no mutation of the conserved Asp entirely prevents folding suggests that there is no obligatory folding order for each repeat and that the folding order is probably not the same for different WD repeat proteins, or even necessarily constant for the same protein.

Amino Acid Sequence↗
PubMed1998

A homology identification method that combines protein sequence and structure information.

A new method is presented for identifying distantly related homologous proteins that are unrecognizable by conventional sequence comparison methods. The method combines information about functionally conserved sequence patterns with information about structure context. This information is encoded in stochastic discrete state-space models (DSMs) that comprise a new family of hidden Markov models. The new models are called sequence-pattern-embedded DSMs (pDSMs). This method can identify distantly related protein family members with a high sensitivity and specificity. The method is illustrated with trypsin-like serine proteases and globins. The strategy for building pDSMs is presented. The method has been validated using carefully constructed positive and negative control sets. In addition to the ability to recognize remote homologs, pDSM sequence analysis predicts secondary structures with higher sensitivity, specificity, and Q3 accuracy than DSM analysis, which omits information about conserved sequence patterns. The identification of trypsin-like serine proteases in new genomes is discussed.

Amino Acid Sequence↗
PubMed1998

A Bayes-optimal sequence-structure theory that unifies protein sequence-structure recognition and alignment.

A rigorous Bayesian analysis is presented that unifies protein sequence-structure alignment and recognition. Given a sequence, explicit formulae are derived to select (1) its globally most probable core structure from a structure library; (2) its globally most probable alignment to a given core structure; (3) its most probable joint core structure and alignment chosen globally across the entire library; and (4) its most probable individual segments, secondary structure, and super-secondary structures across the entire library. The computations involved are NP-hard in the general case (3D-3D). Fast exact recursions for the restricted sequence singleton-only (1D-3D) case are given. Conclusions include: (a) the most probable joint core structure and alignment is not necessarily the most probable alignment of the most probable core structure, but rather maximizes the product of core and alignment probabilities; (b) use of a sequence-independent linear or affine gap penalty may result in the highest-probability threading not having the lowest score; (c) selecting the most probable core structure from the library (core structure selection or fold recognition only) involves comparing probabilities summed over all possible alignments of the sequence to the core, and not comparing individual optimal (or near-optimal) sequence-structure alignments; and (d) assuming uninformative priors, core structure selection is equivalent to comparing the ratio of two global means.

Amino Acid Sequence↗
PubMed1998

Respiratory syncytial virus pneumonitis-induced diffuse alveolar damage in an autologous bone marrow transplant recipient.

A 24-year-old woman with recurrent Hodgkin's lymphoma, stage IIB nodular sclerosing type, underwent an autologous bone marrow transplantation. Forty-five days after transplantation, an upper respiratory tract infection developed that progressed to respiratory distress necessitating mechanical ventilation. An open-lung biopsy demonstrated diffuse alveolar damage. After an extensive search for the cause of the respiratory compromise, we detected respiratory syncytial virus in a bronchoalveolar lavage specimen. The patient was immediately treated with aerosolized ribavirin and intravenous immunoglobulin; her symptoms resolved, and she was extubated 4 days after initiation of therapy.

Adult↗
PubMed1998

Detection of Epstein-Barr virus-specific antibodies by an automated enzyme immunoassay. Performance evaluation and cost analysis.

Detection of antibodies to specific antigens of Epstein-Barr virus (EBV) has been conventionally performed by an immunofluorescence assay (IFA). The procedure is labor intensive and expensive, and interpretation of results is subjective. We evaluated an automated enzyme immunoassay (EIA) (INC-STAR Corp., Stillwater, MN, USA) using 290 serum specimens submitted for the diagnosis of acute infection with EBV. Antibodies (IgG, IgM) to EBV capsid antigen and IgG class antibodies to the nuclear antigen of the virus were obtained using the LABOTECH Automated Microplate Analyzer (BioChem ImmunoSystems Inc., Allentown, PA, USA) and were compared to the antibody profile results obtained by IFA and Western blot as the "gold standard." For detection of acute infection with EBV (presence of IgM and IgG antibodies to the capsid antigen; absence of antibodies to the nuclear antigen), the EIA had 100% sensitivity (11 of 11) and 99% specificity (275 of 279) compared to IFA and Western blot results. A cost analysis of IFA and EIA procedures, based on an estimated annual volume of 12,000 procedures, indicated that $236,000 direct cost and 1,400 h technologist time could be saved with the automated compared with immunofluorescence procedure. The automated EIA for determination of antibodies provides cost-effective, accurate diagnosis of EBV infections in laboratories processing high numbers of specimens now processed by IFA.

Antibodies, Viral↗