Search PubMed⌕ Search

Biomedical subjects

J W Thomas

Publications and source records attributed to J W Thomas.

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

Comparative genome mapping in the sequence-based era: early experience with human chromosome 7.

The success of the ongoing Human Genome Project has resulted in accelerated plans for completing the human genome sequence and the earlier-than-anticipated initiation of efforts to sequence the mouse genome. As a complement to these efforts, we are utilizing the available human sequence to refine human-mouse comparative maps and to assemble sequence-ready mouse physical maps. Here we describe how the first glimpses of genomic sequence from human chromosome 7 are directly facilitating these activities. Specifically, we are actively enhancing the available human-mouse comparative map by analyzing human chromosome 7 sequence for the presence of orthologs of mapped mouse genes. Such orthologs can then be precisely positioned relative to mapped human STSs and other genes. The chromosome 7 sequence generated to date has allowed us to more than double the number of genes that can be placed on the comparative map. The latter effort reveals that human chromosome 7 is represented by at least 20 orthologous segments of DNA in the mouse genome. A second component of our program involves systematically analyzing the evolving human chromosome 7 sequence for the presence of matching mouse genes and expressed-sequence tags (ESTs). Mouse-specific hybridization probes are designed from such sequences and used to screen a mouse bacterial artificial chromosome (BAC) library, with the resulting data used to assemble BAC contigs based on probe-content data. Nascent contigs are then expanded using probes derived from newly generated BAC-end sequences. This approach produces BAC-based sequence-ready maps that are known to contain a gene(s) and are homologous to segments of the human genome for which sequence is already available. Our ongoing efforts have thus far resulted in the isolation and mapping of >3,800 mouse BACs, which have been assembled into >100 contigs. These contigs include >250 genes and represent approximately 40% of the mouse genome that is homologous to human chromosome 7. Together, these approaches illustrate how the availability of genomic sequence directly facilitates studies in comparative genomics and genome evolution.

Animals↗
PubMed1999

The role of focal adhesion kinase binding in the regulation of tyrosine phosphorylation of paxillin.

Focal adhesion kinase (FAK) and paxillin are focal adhesion-associated, phosphotyrosine-containing proteins that physically interact. A previous study has demonstrated that paxillin contains two binding sites for FAK. We have further characterized these two binding sites and have demonstrated that the binding affinity of the carboxyl-terminal domain of FAK is the same for each of the two binding sites. The presence of both binding sites increases the affinity for FAK by 5-10-fold. A conserved paxillin sequence called the LD motif has been implicated in FAK binding. We show that mutations in the LD motifs in both FAK-binding sites are required to dramatically impair FAK binding in vitro. A paxillin mutant containing point mutations in both FAK-binding sites was characterized. The mutant exhibited reduced levels of phosphotyrosine relative to wild type paxillin in subconfluent cells growing in culture, following cell adhesion to fibronectin and in src-transformed fibroblasts. These results suggest that paxillin must bind FAK for maximal phosphorylation in response to cell adhesion and that FAK may function to direct tyrosine phosphorylation of paxillin in the process of transformation by the src oncogene.

Animals↗
PubMed1999

Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase.

Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P. denitrificans oxidase). Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase. The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase. Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center. The data suggest that E89II (E. coli numbering) is critical for the function of the heme copper oxidases. The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant. Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.

Alanine↗
PubMed1999

Fibroblast growth factor-1 (FGF-1) enhances IL-2 production and nuclear translocation of NF-kappaB in FGF receptor-bearing Jurkat T cells.

Fibroblast growth factors (FGFs) are heparin-binding proteins crucial to embryogenesis, angiogenesis, and wound healing. FGF-1 is abundantly expressed in the synovium in rheumatoid arthritis and in rejecting allografts, sites of chronic immune-mediated inflammation. The frequency of FGF-1-responsive T cells is increased in the peripheral blood of these disorders, and a high percentage of infiltrating T cells in rheumatoid arthritis synovium express receptors for FGF-1. To understand the action of FGF-1 in T cells, studies were initiated in Jurkat T cells that express the signaling isoform of FGF receptor-1. These experiments show that FGF-1 stimulation of Jurkat T cells provides a second signal that augments TCR-mediated IL-2 production. Analogous to costimulation via CD28, this activity is mediated through activation of Rel/kappaB, a family of transcription factors known to regulate IL-2 and other activation-inducible proteins. FGF-1 alone induces modest nuclear translocation of kappaB-binding proteins, and this translocation is enhanced by the combination of anti-CD3 and FGF-1. This NF-kappaB binding complex is composed of transcriptionally active p65(RelA)/p50 heterodimers and results primarily from the targeted degradation of IkappaB-alpha, an inhibitor that sequesters Rel/kappaB in the cytoplasm. These data are the first to show a connection between FGF-1 signaling and NF-kappaB activation outside of embryonic development. The signaling events that link FGF receptor-1 engagement and NF-kappaB activation in Jurkat are probably distinct from the CD28 costimulation pathway, since FGF-1-induced Rel/kappaB binding proteins do not contain significant levels of c-Rel and are not identical with the CD28 response complex.

Biological Transport↗
PubMed1999

Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.

Amino Acid Substitution↗
PubMed1999

Infectious complications of 393 peripherally implantable venous access devices in HIV-positive and HIV-negative patients.

PURPOSE: To compare and investigate the rate of infection in patients with and without human immunodeficiency virus (HIV) who have implantable venous access devices placed by interventional radiologists. MATERIALS AND METHODS: Three hundred ninety-one patients undergoing radiologically guided placement of peripheral arm ports were grouped according to their HIV serologic status. Findings were prospectively reviewed in 393 peripherally placed arm ports that were implanted in the basilic, cephalic, or brachial vein under fluoroscopic or sonographic guidance over a 4-year span. Infectious complications were categorized according to severity (local or systemic) and time (periprocedural or late). RESULTS: Three hundred ninety-three ports have been indwelling for a total of 97,256 patient days (range, 1-694; mean duration, 247 days). Among the 30 catheter placements in 29 HIV-positive patients with a total exposure time of 7,242 days, five (one local and four systemic) infections occurred, resulting in a 16.6% overall infection rate, yielding 0.069 infections per 100 catheter days at risk (95% confidence interval [CI], 0.032-0.127). In the remaining 362 HIV-negative patients, 27 (14 local and 13 systemic) infectious complications (7.4%) occurred, translating into 0.030 infections per 100 catheter days (95% CI, 0.021-0.042). The odds ratio of getting an infection from the implantable arm ports in the HIV-positive group was 2.5 times higher than that of the HIV-negative group. The relative risk was similar and was calculated to be 2.3. The P value was .084 (P < .05 required to be considered significant). CONCLUSIONS: These results suggest a significant difference in the infectious complication rate encountered in HIV-positive patients compared with the general population. However, the HIV-positive peripheral arm port infection rate compares favorably with the surgically placed catheters and ports. Many more arm ports in HIV-positive patients must be evaluated for the data to achieve an acceptable level of statistical significance.

Adolescent↗
PubMed1999

Incidence and management of catheter occlusion in implantable arm ports: results in 391 patients.

PURPOSE: To evaluate the incidence and management of catheter occlusion in implantable arm ports. MATERIALS AND METHODS: Findings were prospectively examined in 391 patients in whom 393 arm ports were placed. The indications for port placement included chemotherapy (n = 347), antibiotic administration (n = 35), combination chemotherapy/antibiotic use (n = 7), transfusion (n = 3), and phlebotomy (n = 1). Of the total catheters, 323 (82.2%) underwent tip modification prior to placement. Malfunctioning catheters were usually treated with urokinase instillation. RESULTS: Three hundred ninety-three devices were implanted with 247 mean days of catheter use (total, 97,256 days; range, 1-694 days). The overall incidence of catheter occlusion was 0.14 per 100 catheter days. A single catheter occlusion occurred in 90 (22.9%) catheters, with a mean of 90.1 days before the event. A second occlusion occurred in 36 (9.2%) of the above catheters, with a mean of 60.1 catheter days before the second event. Eighty-five (24.0%) of the 347 cancer patients had at least one occlusive event, yielding a complication rate of 0.098 per 100 catheter days at risk (95% confidence interval [CI]; 0.079-0.114). Of the 35 patients receiving antibiotics, three (8.6%) had at least one occlusive event. This represented a complication rate of 0.032 per 100 catheter days at risk (95% CI; 0.010-0.061). Seventeen (24.3%) of the nonmodified catheters developed an occlusion versus 72 (22.3%) of the modified (P > .05; Fisher exact test). Of the catheters with a first occlusive event, 75 (98.7%) were treated successfully with urokinase instillation. Four (1.0%) patients developed symptomatic subclavian vein thrombosis. No bleeding complications occurred. CONCLUSION: Catheter occlusion is a common complication of long-term arm port placement, with a significantly higher incidence in the cancer patients in our series (P <. 05, Fisher exact test). Catheter tip modification, however, does not considerably affect the incidence of occlusion. Low-dose urokinase therapy is a safe and efficacious treatment of catheter occlusion, obviating the need for catheter removal.

Adolescent↗
PubMed1999

Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1).

Cerebral cavernous malformations (CCM) are congenital vascular anomalies of the brain that can cause significant neurological disabilities, including intractable seizures and hemorrhagic stroke. One locus for autosomal dominant CCM ( CCM1 ) maps to chromosome 7q21-q22. Recombination events in linked family members define a critical region of approximately 2 Mb and a shared disease haplotype associated with a presumed founder effect in families of Mexican-American descent points to a potentially smaller region of interest. Using a genomic sequence-based positional cloning strategy, we have identified KRIT1, encoding a protein that interacts with the Krev-1/rap1a tumor suppressor, as the CCM1 gene. Seven different KRIT1 mutations have been identified in 23 distinct CCM1 families. The identical mutation is present in 16 of 21 Mexican-American families analyzed, substantiating a founder effect in this population. Other Mexican-American and non-Hispanic Caucasian CCM1 kindreds harbor other KRIT1 mutations. Identification of a common Mexican-American mutation has potential clinical significance for presymptomatic diagnosis of CCM in this population. In addition, these data point to a key role for the Krev-1/rap1a signaling pathway in angiogenesis and cerebrovascular disease.

Blood Vessels↗
PubMed1999

Accuracy of risk-adjusted mortality rate as a measure of hospital quality of care.

OBJECTIVES: Reports on hospital quality performance are being produced with increasing frequency by state agencies, commercial data vendors, and health care purchasers. Risk-adjusted mortality rate is the most commonly used measure of quality in these reports. The purpose of this study was to determine whether risk-adjusted mortality rates are valid indicators of hospital quality performance. METHODS: Based on an analytical model of random measurement error, sensitivity and predictive error of mortality rate indicators of hospital performance were estimated. RESULTS: The following six parameters were shown to determine accuracy: (1) mortality risks of patients who receive good quality care and (2) of those who receive poor quality care, (3) proportion of patients (across all hospitals) who receive poor quality care, (4) proportion of hospitals considered to be "poor quality," (5) patients' relative risk of receiving poor quality care in "good quality" and in "poor quality" hospitals, and (6) number of patients treated per hospital. Using best available values for model parameters, analyses demonstrated that in nearly all situations, even with perfect risk adjustment, identifying poor quality hospitals on the basis of mortality rate performance is highly inaccurate. Of hospitals that delivered poor quality care, fewer than 12% were identified as high mortality rate outliers, and more than 60% of outliers were actually good quality hospitals. CONCLUSIONS: Under virtually all realistic assumptions for model parameter values, sensitivity was less than 20% and predictive error was greater than 50%. Reports that measure quality using risk-adjusted mortality rates misinform the public about hospital performance.

Bias↗
PubMed1998

A functional fibroblast growth factor-1 immunoglobulin fusion protein.

Proteins of the fibroblast growth factor (FGF) family play diverse roles in embryonic development, angiogenesis, and wound healing. The most well studied targets of FGF activity typically are cells of mesodermal and neuroectodermal origin; in addition, expression of FGF-1 (acidic FGF) is increased at several sites of chronic immunologic injury, and recent studies show that FGF-1 also may interact with cells of the immune system. In some human T cells, FGF-1 can induce signals necessary for production of interleukin-2, a key cytokine required for T cell proliferation. To better characterize the interaction of FGF-1 with FGF receptors on T cells, a fusion protein was constructed containing a portion of the constant region of human IgG1 (Fc) at the amino terminus of FGF-1. The Fc-FGF-1 fusion protein retained FGF function as determined by stimulation of tyrosine phosphorylation and DNA synthesis in NIH 3T3 cells. Binding of the intact fusion protein to FGF receptor 1 (FGFR1) on T cells was demonstrated by immunoprecipitation of the receptor bound to Fc-FGF-1 and by flow cytometry showing binding of fusion protein to T cells expressing FGFR1. This functional Fc-FGF-1 protein should prove useful in identifying FGFR-expressing cells.

3T3 Cells↗
PubMed1998

Activation by autophosphorylation or cGMP binding produces a similar apparent conformational change in cGMP-dependent protein kinase.

Binding of cyclic nucleotide to or autophosphorylation of cGMP-dependent protein kinase (PKG) activates this kinase, but the molecular mechanism of activation for either process is unknown. Activation of PKG by cGMP binding produces a conformational change in the enzyme (Chu, D.-M., Corbin, J. D., Grimes, K. A., and Francis, S. H. (1997) J. Biol. Chem. 272, 31922-31928; Zhao, J., Trewhella, J., Corbin, J., Francis, S., Mitchell, R., Brushia, R., and Walsh, D. (1997) J. Biol. Chem. 272, 39129-31936). In the present studies, activation of type Ibeta PKG by either autophosphorylation or cGMP-binding alone causes (i) an electronegative charge shift on ion exchange chromatography, (ii) a similar increase ( approximately 3.5 A) in the Stokes radius as determined by gel filtration chromatography, and (iii) a similar decrease in the mobility of the enzyme on native gel electrophoresis. Consistent with these results, cGMP binding increases the rate of phosphoprotein phosphatase-1 catalyzed dephosphorylation of PKG which is autophosphorylated only at Ser-63 (not activated); however, dephosphorylation of PKG that is highly autophosphorylated (activated) is not stimulated by cGMP. The combined results suggest that activation of PKG by either autophosphorylation or cGMP binding alone produces a similar apparent elongation of the enzyme, implying that either process activates the enzyme by a similar molecular mechanism.

Animals↗
PubMed1998

X-ray-induced mutations in mouse embryonic stem cells.

Deletion complexes consisting of multiple chromosomal deletions induced at single loci can provide a means for functional analysis of regions spanning several centimorgans in model genetic systems. A strategy to identify and map deletions at any cloned locus in the mouse is described here. First, a highly polymorphic, germ-line competent F1(129/Sv-+Tyr+p x CAST/Ei) mouse embryonic stem cell line was established. Then, x-ray and UV-induced mutagenesis was performed to determine the feasibility of generating deletion complexes throughout the mouse genome. Reported here are the selection protocols, induced mutation frequencies, cytogenetic and extensive molecular analysis of mutations at the X-chromosome-linked hypoxanthine phosphoribosyltransferase (Hprt) locus and at the neural cell adhesion molecule (Ncam) locus located on chromosome 9. Mutation analysis with PCR-based polymorphic microsatellite markers revealed deletions of <3 cM at the Hprt locus, whereas results consistent with deletions covering >28 cM were observed at the Ncam locus. Fluorescence in situ hybridization with a chromosome 9 paint revealed that some of the Ncam deletions were accompanied by complex chromosome rearrangements. In addition, deletion mapping in combination with loss of heterozygosity of microsatellite markers revealed a putative haploinsufficient region distal to Ncam. These data indicate that it is feasible to generate x-ray-induced deletion complexes in mouse embryonic stem cells.

Animals↗
PubMed1998

SH2- and SH3-mediated interactions between focal adhesion kinase and Src.

Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2- and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAKPro-2) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.

Amino Acid Sequence↗
PubMed1998

Interpreting risk-adjusted length of stay patterns for VA hospitals.

OBJECTIVES: The Veterans Health System must become more competitive with the private sector in terms of efficiency of care. Studies have shown significantly longer lengths-of-stay (LOS) in facilities operated by the Department of Veterans Affairs (VA) compared with private sector facilities. Most comparisons, however, have not controlled well for casemix differences or have involved small numbers of patients. The aims of this study were: (1) controlling for casemix, to accurately measure the degree by which average length of stay in Veterans Affairs facilities exceeds that of private sector hospitals and (2) to demonstrate a methodology with which individual VA facilities can identify clinical and demographic subgroups of patients associated with the higher length-of-stay averages. METHODS: Subjects of the study were Veterans Health System patients hospitalized during 1991-1993 and veteran respondents to the 1991 National Hospital Discharge Survey. Hospitals' mean length of stay adjusted for patients' diagnosis related groups, severity, demographics, and travel distances were measured. RESULTS: Veterans Affairs medical centers' average risk-adjusted length of stay was 36% higher (8.9 days compared with 6.5 days) than that of the private sector. For individual hospitals, relative length-of-stay efficiency typically varied by condition. Among 14 hospitals in the VA's midwest region, none were high risk-adjusted length-of-stay outliers in all conditions studied, and four were high outliers for some conditions and low outliers for others. CONCLUSIONS: Controlling for differences in patient demographic and clinical factors, Veterans Affairs medical centers consumed significantly more days of care than private sector hospitals. Veterans Affairs medical centers will be able to improve efficiency by identifying specific subgroups of patients whose clinical treatment should be examined.

Comorbidity↗