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Biomedical subjects

T Mitchell

Publications and source records attributed to T Mitchell.

At least 19 recordsLinked to original sources
PubMed1992

A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae.

We report the discovery of a group of highly conserved DNA sequences located, in those cases studied, within intergenic regions of the chromosome of the Gram positive Streptococcus pneumoniae. The S. pneumoniae genome contains about 25 of these elements called BOX. From 5' to 3', BOX elements are composed of three subunits (boxA, boxB, and boxC) which are 59, 45 and 50 nucleotides long, respectively. BOX elements containing one, two and four copies of boxB have been observed; boxB alone was also detected in one instance. These elements are unrelated to the two most thoroughly documented families of repetitive DNA sequences present in the genomes of enterobacteria. BOX sequences have the potential to form stable stem-loop structures and one of these, at least, is transcribed. Most of these elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of S. pneumoniae. This location raises the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence-specific and virulence-related genes.

Base Sequence↗
PubMed1992

Localization of tumor necrosis factor receptors in the synovial tissue and cartilage-pannus junction in patients with rheumatoid arthritis. Implications for local actions of tumor necrosis factor alpha.

OBJECTIVE: We have previously described the location of tumor necrosis factor alpha (TNF alpha)-producing cells in synovial tissue and cartilage-pannus junction in rheumatoid arthritis (RA). To further understand the local actions of TNF alpha, we investigated the expression of TNF receptors (TNF-R) on cells in the same compartments in patients with RA. METHODS: The expression of both p55 TNF-R and p75 TNF-R was determined using alkaline phosphatase-conjugated mouse anti-alkaline phosphatase (APAAP) and double immunofluorescence staining techniques with monoclonal antibodies. RESULTS: In RA synovial membrane, both p55 TNF-R and p75 TNF-R were detectable in up to 90% of the cells in the lining layer, and were demonstrated on cells in deeper layers of the membrane, including vascular endothelial cells. Cells in lymphoid aggregates expressed both TNF-R, but with a predominant expression of p75 receptor. At the cartilage-pannus junction, the majority of pannus cells, especially those invading cartilage, expressed both the p55 and the p75 TNF-R. Sequential section and double immunofluorescence staining showed that the TNF-R-expressing cells were in the vicinity of TNF alpha-containing cells, and some TNF alpha-containing cells also expressed TNF-R. TNF-R-expressing cells were also detected in osteoarthritic and normal synovial tissue, but in smaller numbers and at a lower intensity. CONCLUSION: These results provide histologic evidence that both p55 TNF-R and p75 TNF-R are expressed by a variety of cell types in RA synovial tissue, reflecting the fact that a wide range of cells are potential targets for TNF alpha in this tissue. This study further supports the hypothesis that TNF alpha plays a major role in the pathogenesis of RA.

Arthritis, Rheumatoid↗
PubMed1992

Enhanced expression of tumor necrosis factor receptor mRNA and protein in mononuclear cells isolated from rheumatoid arthritis synovial joints.

We previously proposed the hypothesis that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA) based on our observations that it is the dominant inducer of interleukin-1 (IL-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in RA synovial joint mononuclear (MNC) cells in culture. Since TNF-alpha acts via two membrane receptors, we have extended those studies to investigate the distribution of the p55 and p75 TNF receptors (TNF-R) in RA tissue. Surface receptor expression was quantitated by flow cytometry using monoclonal antibodies specific to the p55 (HTR-9) and the p75 (UTR-1) TNF-R. Both receptors were significantly increased on MNC isolated from the synovial membrane of RA patients compared to normal or RA peripheral blood MNC. Interestingly, the p75 TNF-R was increased both on large monocytic/macrophage-type cells and CD3+ lymphocytes. Furthermore, there was a significant increase in the proportion of CD3+ cells in RA synovial fluid expressing the p75 TNF-R, compared to matched peripheral blood MNC. In contrast to RA synovial MNC, p75 or p55 TNF-R expression was not significantly increased in osteoarthritis synovial MNC. In addition, Northern blot analysis indicated abundant expression of both p55 and p75 mRNA in RA synovial joint MNC. This was in contrast to normal peripheral blood MNC cells which contained little or no constitutive TNF-R mRNA; following stimulation with phytohemagglutinin and IL-2, a rapid and transient expression of both receptor mRNA was induced. These results, therefore, indicate that in RA synovial joint tissue there is up-regulation of both p55 and p75 TNF-R mRNA and surface protein expression, and with the presence of TNF-alpha in RA tissues, these results provide support to our hypothesis that TNF-alpha is of critical importance in the pathogenesis of RA.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Interference established in mice by infection with Friend murine leukemia virus.

Retroviral interference is manifested in chronically infected cells as a decrease in susceptibility to superinfection by virions using the same cellular receptor. The pattern of interference reflects the cellular receptor specificity of the chronically infecting retrovirus and is mediated by the viral envelope glycoprotein, which is postulated to bind competitively all cellular receptors available for viral attachment. We established retroviral interference in mice by infecting them with Friend murine leukemia virus and them measured susceptibility to superinfection by challenging the mice with the erythroproliferative spleen focus-forming virus. Infection of approximately 10% of nucleated splenocytes rendered mice 1% as susceptible to superinfection as untreated controls. The magnitude of this effect was the same in mice incapable of producing neutralizing antibodies or genetically deficient for T cells. The results indicated that retroviral interference in vivo was established rapidly with infection of a fraction of the host cell population and that the decrease in susceptibility to superinfection occurred without a detectable contribution by immunologic factors.

Animals↗
PubMed1992

The alternation between atrial flutter and atrial fibrillation.

Atrial fibrillation and atrial flutter share a common reentrant mechanism. However, the relationship between these arrhythmias has not been systemically studied to date. To evaluate the degree to which these arrhythmias may alternate, consecutive Holter monitor recordings which showed fibrillation or flutter in 96 patients were reviewed. One half of the patients were studied after open-heart surgery and the other half for varying indications. One quarter of the patients had atrial flutter in addition to fibrillation, and this alternation with flutter was significantly associated with the use of a type 1A antiarrhythmic drug (p = 0.007), but not with the use of digoxin or beta blockers (p = NS for both). Furthermore, this alternation with flutter was more common in the postoperative group (p = 0.01). A history of embolization was less common in patients who were in the postoperative group (p = 0.003) and patients who had flutter in addition to fibrillation (p = 0.05).

Adolescent↗
PubMed1991

Induction of plasmacytomas secreting antigen-specific monoclonal antibodies with a retrovirus expressing v-abl and c-myc.

ABL-MYC, a recombinant murine retrovirus that expresses v-abl and c-myc, rapidly induces transplantable mono- or oligoclonal plasmacytomas in BALB/c mice. To determine if the targets for transformation of this retrovirus are antigen-committed B lymphocytes and to explore this system as an alternative technique for producing antigen-specific monoclonal antibodies, plasmacytomas were induced in mice that had been immunized with two different types of immunogens, hen egg white lysozyme and sheep red blood cells. The majority of these plasmacytomas secreted immunogen-specific antibodies. Plasmacytomas induced in unimmunized mice did not react with hen egg white lysozyme or sheep red blood cells. The specific antibodies were comparable in concentration, specificity, and affinity to monoclonal antibodies obtained with conventional hybridoma technology, but, in addition to IgGs and IgMs, they included specific IgA antibodies, which are rare among splenic-derived hybridomas. Our results demonstrate that a principal target for ABL-MYC is an antigen-committed B lymphocyte. In addition this procedure provides an alternative method for the production of monoclonal antibodies, without a requirement for hetero-caryon formation by cell fusion techniques.

Animals↗
PubMed1991

Phosphorylation and formation of hybrid enzyme species test the "half of sites" reactivity of Escherichia coli succinyl-CoA synthetase.

Recent sequencing experiments have identified alpha-His246 as the phosphorylation site of Escherichia coli succinyl-CoA synthetase [Buck, D., Spencer, M. E., & Guest, J. R. (1985) Biochemistry 24, 6245-6252]. We have replaced alpha-His246 with an asparagine residue using site-directed mutagenesis techniques. The resulting mutant enzyme (designated H246N) exhibited no enzyme activity, as expected, but was found as a structurally intact, stable tetramer. Small differences in the net charge of H246N and wild-type enzymes were first detected on native polyacrylamide gels. These charge differences were resolved by using native isoelectric focusing gels to further separate the wild-type enzyme into diphosphorylated, monophosphorylated, and unphosphorylated species. The enzyme species were found to be interconvertible upon incubation with the appropriate enzyme substrate(s). Sample mixtures containing increasing molar ratios of H246N (alpha H246N beta)2 to wild-type enzyme (alpha beta)2 were unfolded and then refolded. The refolded enzyme mixtures were analyzed for enzymatic activity and separated on native isoelectric focusing gels. The hybrid enzyme (alpha beta alpha H246N beta) retained a significant amount of enzyme activity and also exhibited substrate synergism (stimulation of succinate in equilibrium succinyl-CoA exchange in the presence of ATP). Substrate synergism with this enzyme has been interpreted as evidence for interaction between active sites in such a way that only a single phosphoryl group is covalently attached to the enzyme at a given time [Wolodko, W. T., Brownie, E.R., O'Connor, M. D., & Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119]. On the contrary, we conclude that tetrameric succinyl-CoA synthetase from E. coli is comprised of two independently active dimer molecules associated together to form a "dimer of dimers" that displays substrate synergism within each dimer and not necessarily between dimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Intrinsic fluorescence of succinyl-CoA synthetase and four tryptophan mutants. Tryptophan 76 and tryptophan 248 of the beta-subunit are responsive to CoA binding.

Previous studies showed that modification of an average of one of the three tryptophan residues of succinyl-CoA synthetase of Escherichia coli abolished enzyme activity, but did not prevent phosphorylation of the enzyme by ATP [Ybarra, J., Prasad, A. R. S., & Nishimura, J.S. (1986) Biochemistry 25, 7174-7178]. In the present study, single mutations in which each of the three tryptophans (beta-Trp43, beta-Trp76, and beta-Trp248) has been changed to phenylalanine (designated W43F, W76F, and W248F) have been accomplished by the technique of site-directed mutagenesis and the mutant proteins isolated. In addition, a double mutant in which beta-Trp43 and beta-Trp248 were changed to phenylalanines (W43,248F) has also been isolated. Each of the mutant enzymes was practically as active as wild type. Since the emission spectrum of beta-Trp76 reflected a low fluorescence intensity for this residue, it was possible to obtain the emission spectrum of each tryptophan residue by using W43F, W248F, and W43,248F. From the positions of the emission maxima and the results of iodide quenching of fluorescence, it was deduced that beta-Trp248 is a surface residue, beta-Trp43 is buried, and beta-Trp76 is intermediate in location. Coenzyme A, but no other substrate, protected the fluorescence of beta-Trp76 and beta-Trp248, but not of beta-Trp43, against quenching by acrylamide. These results are consistent with an interaction between beta-Trp76 and beta-Trp248 and the binding site for CoA.

Adenosine Diphosphate↗
PubMed1989

Infusion of vincristine and doxorubicin with oral dexamethasone as first-line therapy for multiple myeloma.

32 previously untreated patients with multiple myeloma received vincristine, doxorubicin ('Adriamycin'), and dexamethasone (VAD) as first-line therapy. The overall response rate was 84%, with 28% of all patients entering complete remission. Response was rapid, with near-maximum response occurring after two courses of treatment and rapid improvement in bone-marrow function. Median response duration was 18 months and this seemed to be unaffected by initial prognostic criteria or by degree of remission achieved. The projected median survival was 44 months, with 75% of all patients and 83% of responders being alive at 2 years. Side-effects due to steroids were common, but there was only 1 treatment-related death. The high response rate and lack of toxicity offer an advantage over other forms of initial treatment, although other strategies will be necessary to prolong the duration of response.

Administration, Oral↗
PubMed1989

Ocular blood flow in dogs using radiolabelled microspheres.

We used radioactive microspheres of different sizes to determine the rate of blood flow through the various parts of the eye in dogs. The use of either 15 +/- 5 microns or 25 +/- 5 microns microspheres gave reproducible measurements of total ocular and uveal blood flows. Using these sphere sizes, retinal flow was found to be close to 1% of the total ocular blood flow and showed poor reproducibility due in part to the small number of spheres trapped in the retinal vessels.

Animals↗
PubMed1989

Effect of Streptococcus pneumoniae on human respiratory epithelium in vitro.

A total of 11 of 15 Streptococcus pneumoniae culture filtrates and all five bacterial autolysates produced by cell death in the stationary phase caused slowed ciliary beating and disruption of the surface integrity of human respiratory epithelium in organ culture. This effect was inhibited by cholesterol and was heat labile and reduced by standing at room temperature but was stable at -40 degrees C. The activity was detected at the late stationary phase of culture and was associated with the presence of hemolytic activity. Gel filtration of a concentrated culture filtrate and autolysate both yielded a single fraction of approximately 50 kilodaltons which slowed ciliary beating and were the only fractions with hemolytic activity. Rabbit antiserum to pneumolysin, a sulfhydryl-activated hemolytic cytotoxin released by S. pneumoniae during autolysis, neutralized the effect of the culture filtrate on respiratory epithelium. Both native and recombinant pneumolysin caused ciliary slowing and epithelial disruption. Electron microscopy showed a toxic effect of pneumolysin on epithelial cells: cytoplasmic blebs, mitochondrial swelling, cellular extrusion, and cell death, but no change in ciliary ultrastructure. Recombinant pneumolysin (10 micrograms/ml) caused ciliary slowing in the absence of changes in cell ultrastructure. Release of pneumolysin in the respiratory tract during infection may perturb host defenses, allowing bacterial proliferation and spread.

Autolysis↗
PubMed1987

Expression of the c-raf protooncogene in human hematopoietic cells and cell lines.

The murine sarcoma virus 3611 contains the transforming v-raf gene that has partial nucleotide homology with the src family of tyrosine kinase-encoding oncogenes. Although this virus induces fibrosarcomas in mice, a recombinant murine retrovirus carrying both the raf and myc oncogenes induces immunoblastic lymphomas and immortalizes mouse macrophages in vitro. The present study has thus monitored the expression of c-raf in human hematopoietic cells. The results demonstrate the presence of a 3.6-kb c-raf transcript in HL-60 promyelocytic leukemic cells. The induction of HL-60 cell differentiation along the monocytic or granulocytic lineages had no detectable effect on the level of c-raf transcripts. Furthermore, in contrast to c-myc and c-fms expression, inhibition of protein synthesis with cycloheximide had no detectable effect on c-raf expression. Similar levels of c-rafRNA were also found in other human cell lines derived from myeloid, B cell, and T cell tumors, as well as in normal granulocytes, monocytes, and macrophages. These findings suggest that the c-raf protooncogene is widely expressed in multiple hematopoietic lineages.

Cell Differentiation↗
PubMed1977

Plasma uric acid and plasma albumin in healthy subjects.

In healthy male subjects there was a positive correlation between plasma uric acid and plasma albumin (r = 0 - 43, P less than 0 - 005, n = 49) when repeated measurements of both variables were used for each subject. Changes in plasma albumin induced by in vivo ultrafiltration were not accompanied by changes in plasma uric acid. The correlation of plasma uric acid with plasma albumin cannot be attributed to protein binding of urate. The two variables are probably related indirectly through a common association with an unknown factor or factors.

Adolescent↗