Biomedical subjects

M R Smith

Publications and source records attributed to M R Smith.

At least 73 records · Page 4Linked to original sources

PubMed1998

Overexpression of phosphoinositide-specific phospholipase Cgamma in NIH 3T3 cells promotes transformation and tumorigenicity.

Phosphoinositide-specific phospholipase Cgamma (PLCgamma) is a key regulatory enzyme that binds to the phosphoryl-tyrosine residues in the cytoplasmic domain of certain activated receptors and catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] forming IP3 and diacylglycerol (DAG) in response to several mitogenic factors. Previously, we determined that microinjected PLCgamma induces DNA synthesis in G0-arrested NIH 3T3 cells, suggesting the possibility that PLCgamma may have an oncogenic potential. In this report, we demonstrate that overexpression of PLCgamma in NIH 3T3 cells results in altered growth properties and cellular transformation. The PLCgamma/3T3 transfectants do not require serum growth factors to proliferate, display anchorage-independent growth in soft agar and induce tumors when transplanted into nude mice. These findings suggest that overexpression of PLCgamma facilitates the transformation of NIH 3T3 cells. Furthermore, PLCgamma expression and activity have been shown to be elevated in many human tumors. Thus, PLCgamma signaling may contribute to the promotion and/or progression of human cancers.

3T3 Cells↗

PubMed1998

Enhancement of Exopolysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 with a Simplified Defined Medium.

The aim of this work was to investigate the medium requirements for growth and production of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772. The strain was grown in batch cultures on a chemically defined medium, and the technique of single omission of medium components was applied to determine the nutritional requirements. The omission of aspartic acid, glutamic acid, or glycine affected growth only slightly, and the omission of glutamine, asparagine, or threonine resulted in a stronger reduction of the growth. All the other amino acids were essential. Multiple omissions of amino acids caused an almost complete loss of growth. L. delbrueckii subsp. bulgaricus required only riboflavin, calcium pantothenate, and nicotinic acid as individual vitamins. Surprisingly, when only these vitamins were present in the medium and other vitamins were not, less growth was observed than in the complete medium but the amount of exopolysaccharide produced was significantly greater. These observations were studied in more detail with a simplified defined medium in which L. delbrueckii subsp. bulgaricus was able to grow and produce exopolysaccharides. Although the final optical density in the simplified medium was lower, the production of exopolysaccharides was about twofold higher than in the complete medium.

Journal Article↗

PubMed1998

Serum CRP and IL-6 levels after trauma. Not predictive of septic complications in 31 patients.

We studied 31 blunt trauma victims, Injury Severity Score (ISS) mean 14 (9-57), for the pattern of release of C-reactive protein (CRP) and cytokine interleukin-6 (IL-6). Blood samples were taken on admission (within 6 hours of injury), as well as at 24 hours, and 3, 5 and 7 days. Serum CRP and IL-6 were measured by ELISA. Subsequent surgical events and sepsis were noted. Serum IL-6 levels on admission were considerably higher (median 135 pg mL-1) than our laboratory reference range (< 5 pg mL-1), slowly returning towards reference values during the study. Serum CRP levels were similar to laboratory normal values on admission (median 8.5 mg L-1 vs 7.5 mg L-1), reaching peak values (median 110 mg L-1) after 3 days. There was a correlation between IL-6 release and ISS but not between CRP and ISS. Patients undergoing surgery showed further increases in IL-6 and CRP levels postoperatively. Of 24 surgical patients, 9 developed postoperative sepsis. In blunt trauma patients, early assessment of the markers CRP or IL-6 were not useful for the diagnosis of sepsis. Levels of CRP following accidental or surgical trauma should be assessed with caution.

Adult↗

PubMed1998

Bryostatin 1 down-regulates mdr1 and potentiates vincristine cytotoxicity in diffuse large cell lymphoma xenografts.

The down-regulation of multidrug resistance (mdr1) gene expression as detected by competitive reverse transcription-PCR and the antitumor activity of bryostatin 1 (Bryo1) are investigated in a newly established cell line from a patient with relapsed diffuse large cell lymphoma (DLCL). The cell line (WSU-DLCL2) grows in liquid culture and forms s.c. tumors in mice with severe combined immune deficiency. WSU-DLCL2 is a mature B-cell line (IgG lambda) that is negative for EBV nuclear antigen, expresses the multidrug resistance phenotype, and has t(14;18)(q32;q21) plus other chromosomal aberrations. Exposure of the WSU-DLCL2 cells to Bryo1 in culture reversed the multidrug resistance phenotype within 24 h. A functional assay revealed a 4-fold increase in [3H]vincristine accumulation in Bryo1-treated cells compared with control. Vincristine (VCR), doxorubicin, Bryo1, and 1-beta-D-arabinofuranosylcytosine showed no clinically significant activity when given alone to WSU-DLCL2-bearing severe combined immune deficiency mice. However, when given 24 h before each cytotoxic agent, Bryo1 substantially increased the antitumor activity of VCR but not 1-beta-D-arabinofuranosylcytosine. There was a statistically significant (P < 0.001) decrease in the expression of P-glycoprotein in WSU-DLCL2 tumors taken from Bryo1-treated animals compared with untreated controls. In vivo, a competitive reverse transcription-PCR assay revealed decreased mdr1 RNA expression 24 h after Bryo1 treatment. These findings taken together indicate that Bryo1-induced down-regulation of mdr1 might be one mechanism by which Bryo1 potentiates VCR activity. The sequential use of both agents resulted in clinically significant antitumor activity in this lymphoma model.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

IL-7 activates alpha4beta1 integrin in murine thymocytes.

IL-7, a cytokine produced by thymic epithelium, was shown to induce adhesion of murine thymocytes to gelatin-coated membranes. A major binding component of gelatin was identified as fibronectin. IL-7-induced adhesion was observed for all of the major thymocyte subsets, including double-negative, double-positive, and single-positive cells, and specific IL-7R were verified on each subset. Fibronectin binding was mediated via alpha4beta1 integrin (VLA-4), which is expressed at high levels on thymocytes. VLA-4 surface expression was not increased following IL-7 treatment, but was shown to undergo rapid tyrosine phosphorylation on the beta1 subunit. This tyrosine phosphorylation was blocked by genistein, which also blocked IL-7-induced adhesion. IL-7 was detected on the extracellular matrix of the thymus, suggesting that it could promote matrix association through an integrin pathway.

Animals↗

PubMed1997

Microinjected cDNA encoding JAK2 protein-tyrosine kinase induces DNA synthesis in NIH 3T3 cells.

Microinjection of expression plasmids encoding either JAK2 or hyperactive (Ndelta661)rJAK2 into serum-starved NIH 3T3 cells resulted in 20-30-fold induction of DNA synthesis. Control microinjections of buffer or parental pcDNA3 vector resulted in only 3-5-fold induction of DNA synthesis. Induction of DNA synthesis was blocked when plasmid encoding JAK2 was microinjected in the presence of the JAK2-selective inhibitor AG-490, whereas AG-490 did not block DNA synthesis induced by microinjected plasmid encoding (Ndelta661)rJAK2. The ability of JAK2 to initiate the G(o)/S cell cycle transition is comparable to that of other proto-oncogenes, and supports a mechanistic role for overexpressed Janus kinases in carcinogenesis.

3T3 Cells↗

PubMed1997

Malignant transformation of mammalian cells initiated by constitutive expression of the polo-like kinase.

Polo-like kinase (Plk) is the mammalian homologue of the Drosophila polo and Saccharomyces cerevisiae CDC5 genes, which are thought to be involved in regulating chromosomal segregation. Previously, we showed that transient ectopic expression of Plk could induce DNA synthesis in quiescent NIH 3T3 cells, suggesting that Plk might also have a function during G1 or S phase. Here we report that microinjection of Plk mRNA is sufficient to drive quiescent cells into mitosis and that constitutive expression of Plk in NIH 3T3 cells causes oncogenic focus formation. These transformed cells grow in soft agar and form tumors in nude mice. Because Plk expression has been shown to be high in various human tumors, we suggest that Plk may contribute to the promotion and/or progression of human cancers.

3T3 Cells↗

PubMed1997

TIMP-3 induces cell death by stabilizing TNF-alpha receptors on the surface of human colon carcinoma cells.

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) regulate the structural integrity of the extracellular matrix (ECM). Constitutive expression of human TIMP-3 in human DLD colon carcinoma cells renewed serum-responses and inhibited tumour formation in nude mice. To elucidate the mechanism of TIMP-3-mediated tumour suppression, we compared parental DLD and TIMP-3 expressing DLD cells (TIMP-3/DLD), finding them to be significantly different. TIMP-3/DLD cultures have fewer mitotic cells, are delayed in G1, and die after serum starvation. TIMP-3/DLD conditioned media activates cell death on fibroblast cells. The cell death induced by serum starvation and conditioned media was inhibited by 70%, in the presence of neutralizing tumour necrosis factor alpha (TNF-alpha) antibody. TIMP-3/DLD whole cell lysate contained p55 TNF-alpha receptor, while vector/DLD lysate had p55 TNF-alpha receptor and p46 soluble TNF-alpha inhibitor. Vector/DLD conditioned media had p46, while no soluble TNF-alpha receptor was detected in TIMP-3/DLD conditioned media. In addition, FACS analysis revealed that TIMP-3/DLD cells have more TNF-alpha surface binding sites, suggesting a direct correlation between TIMP-3 expression and surface receptors. The mechanism of tumorigenic reversion induced by TIMP-3 in DLD cells may involve protection of receptors from the proteolytic activity of MMPs. Putative TIMP-3-mediated inhibition of MMPs restores the TNF-alpha p55 signalling pathway and the carcinoma cell is killed by autocrine TNF-alpha. Thus, DLD cells have specific ECM MMPs that cleave cytokines and cytokine receptors. TIMP-3 specifically inhibits MMPs involved in receptor shedding.

Antigens, CD↗

PubMed1997

Colposacropexy: an alternative technique.

Colposacropexy procedures restore anatomically correct apical vaginal support on the levator plate at the ischial spine level. Venous hemorrhage resulting from laceration of presacral veins during suture fixation is the major hazard of this procedure. Titanium orthopedic bone anchor fixation minimizes this risk through precision placement of the bone anchor-suture unit.

Female↗

PubMed1997

Interaction between the sodium channel inactivation linker and domain III S4-S5.

The III-IV linker (L(III-IV)) of the rat brain sodium channel is critical for fast inactivation, possibly forming a fast inactivation particle. Inactivation can be disrupted by mutation of a conserved alanine at position 1329 in the S4-S5 loop of domain III. Combination of a charged mutation at 1329 with a compensatory (opposite) charge mutation at position 1489 in L(III-IV) partially restores inactivation of the channel. The compensatory charge mutant channel has a single-channel mean open time that is similar to that of the wild-type channel and is approximately 50 times shorter than that of the L(III-IV) mutant channel. The results of thermodynamic cycle analysis indicate that the mutations in domain III S4-S5 and L(III-IV) have a coupling energy of 2.8 kcal/mol, indicating that the two mutations act interdependently. These data suggest that L(III-IV) interacts directly with A1329, which may form part of the docking site if L(III-IV) is a fast inactivation particle.

Action Potentials↗

PubMed1997

The value of transformed lymphocyte count in subclassification of non-Hodgkin's lymphoma by fine-needle aspiration.

No established criteria exist for predicting lymphoma grade or transformation in cytologic material. We counted transformed lymphocytes in fine-needle aspiration (FNA) biopsy specimens to determine whether the percentage of these cells in the smear could predict the histologic grade, the biologic behavior, or both. The percentage of transformed lymphocytes out of total lymphoid cells was determined on Papanicolaou-stained smears. Afterward, a cytodiagnosis was based on clinical information available at the time of the FNA, cytomorphologic data, and flow cytometry data. Results were correlated with results of examination of the surgical biopsy specimen, clinical behavior of the lymphoma, or both. The percentage of transformed lymphocytes was 10% or less in all low-grade or indolent lymphomas. Aspirates with transformed lymphocyte counts of 20% or greater were aggressive lymphomas. We also report our experience in the diagnosis of non-Hodgkin's lymphoma by FNA using cytomorphologic examination and immunophenotyping by flow cytometry at a cancer referral hospital. This is a preliminary study, and larger series may help establish the ranges of transformed lymphocyte counts that correlate with the lymphoma subtype.

Biopsy, Needle↗

PubMed1997

Leuconostoc mesenteroides B-1355 Mutants Producing Alternansucrases Exhibiting Decreases in Apparent Molecular Mass.

Mutants of Leuconostoc mesenteroides B-1355 exhibiting decreases in the apparent molecular mass of alternansucrase on sodium dodecyl sulfate (SDS)-polyacrylamide gels stained for enzyme activity were isolated after mutagenizing strain R15 with N-methyl-N(prm1)-nitro-N-nitrosoguanidine. Strain R15 was a UV mutant of strain B-1355 which was enriched for production of alternansucrase. All strains produced principal and minor alternansucrase bands on SDS gels when cultures were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of the principal and minor activity bands on our SDS gels did not result from dextran-enzyme complexes, because mutants constitutive for synthesis of glucosyltransferases (GTFs) on sugars other than sucrose produced activity bands after growth in glucose medium that were the same as those produced after growth in sucrose medium. Dextransucrase, which had been inactivated by heating at 45(deg)C, was reactivated when subjected to SDS-PAGE, showing that our SDS-PAGE conditions were reversibly denaturing. Thermal denaturation at 45(deg)C did not involve a dispersal of GTFs into subunits. Densitometry measurements showed a roughly linear relationship between enzyme activity and band intensity over a loading range of 0.2 to 0.8 mU per sample well. We concluded that SDS-PAGE followed by activity staining was a reliable method for estimating numbers and ratios of GTFs produced by Leuconostoc sp. in media containing sucrose.

Journal Article↗

PubMed1997

Genetic determinant of rapid-onset B-cell lymphoma by avian leukosis virus.

Infection of 10 day-old chicken embryos with the recombinant avian leukosis virus (ALV) EU-8 induces a high incidence of rapid-onset B-cell lymphoma by insertional activation of the c-myb gene. LR-9, a related ALV with differences from EU-8 in the gag and pol genes, induces rapid-onset lymphoma at only a low incidence. To localize the viral determinant(s) responsible for this biologic difference, we constructed and tested a series of reciprocal chimeras between EU-8 and LR-9 ALVs. The ability to induce rapid-onset lymphoma efficiently was localized to a 925-nucleotide (nt) region of the EU-8 gag gene. Sequence analysis of the region revealed a 42-nt deletion in EU-8 relative to LR-9, as well as some single-nucleotide changes. A mutant virus, delta LR-9, constructed by deleting these 42 nt from LR-9, also induced rapid-onset lymphoma at a high frequency, confirming the biologic significance of this deletion. This deletion removed nt 735 to 776, which lies within a cis-acting RNA element that negatively regulates splicing (NRS). The deletion was shown to cause an increase in splicing efficiency, which may lead to increased production of a truncated myb gene product from an ALV-myb readthrough RNA.

Animals↗

PubMed1997

Rapid induction of B-cell lymphomas by avian leukosis virus.

Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene and produce an ALV-myb read-through RNA, which is spliced to produce a truncated Myb protein. The genetic determinants of such recombinant ALVs have been mapped to a 42-nt deletion within the gag gene. This deletion increases splicing efficiency since it is located within a negative regulator of splicing. We propose that the deletion leads to increased production of Myb protein by increasing splicing of an ALV-myb pre-mRNA.

Animals↗

PubMed1997

[Superior vena cava and right atrium thrombosis successfully treated with streptokinase].

The case of a 56 year-old male with acute lymphoid leukemia and no signs of activity for the last four months is reported. He presented with superior vena cava thrombosis caused by a Hickman catheter, and had positive blood cultures for Candida albicans and Staphylococcus epidermidis. Despite adequate antimicrobial therapy, the fever persisted, and the patient was submitted to surgical thrombectomy. One week following the procedure, the fever returned, and thrombosis of the superior vena cava extending to the right atrium was identified by transesophageal echocardiography (TEE). The patient underwent thrombolytic therapy with streptokinase, and no thrombus could be identified in the control TEE. No hemorrhagic or thromboembolic complication occurred. The patient was discharged with oral anticoagulation.

Catheters, Indwelling↗

PubMed1996

A missense mutation in the sodium channel Scn8a is responsible for cerebellar ataxia in the mouse mutant jolting.

The voltage-gated sodium channel Scn8a is broadly distributed in brain and spinal cord. We have identified a missense mutation in Scn8a that is associated with cerebellar ataxia in the jolting mutant, a mild allele of the "motor endplate disease" locus. The jolting mutation results in substitution of Thr for an evolutionarily conserved Ala residue in the cytoplasmic S4-S5 linker of domain III. Introduction of the corresponding mutation into the rat brain IIA sodium channel shifted the voltage dependence of activation by 14 mV in the depolarizing direction, without affecting the kinetics of fast inactivation or recovery from inactivation. A shift in the threshold of the Scn8a channel could account for the reduced spontaneous activity of Purkinje cells, reduced inhibitory output from the cerebellum, and loss of motor control observed in jolting mice.

Amino Acid Sequence↗

PubMed1996

A bacterial invasin induces macrophage apoptosis by binding directly to ICE.

Shigella, the etiological agent of dysentery, kills macrophages by inducing apoptosis. Deletion mutants in the invasion invasion plasmid antigen B (ipaB) of Shigella flexneri are not cytotoxic. Here, we localized IpaB to the cytoplasm of macrophages infected with S. flexneri. Purified IpaB induced apoptosis when microinjected into macrophages, indicating that IpaB is sufficient to induce apoptosis. Using a GST-IpaB fusion protein as a ligand in affinity purification, we isolated four IpaB binding proteins from macrophages which were identified as the precursor and the mature polypeptides of interleukin-1beta converting enzyme (ICE) or a highly homologous protease. We found that IpaB binds directly to ICE and this enzyme is activated during S. flexneri infection. Furthermore, specific inhibitors of ICE prevented Shigella-induced apoptosis.

Adhesins, Bacterial↗

PubMed1996

PLC gamma 1 Src homology domain induces mitogenesis in quiescent NIH 3T3 fibroblasts.

Previously, we demonstrated that microinjection of phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1) and lipase-defective mutants of PLC gamma 1 induced G(0) growth arrested NIH 3T3 fibroblasts to enter S phase of the cell cycle. These experiments suggested that regions other than the catalytic domain of PLC gamma 1 may be responsible for inducing mitogenesis. To test other regions of PLC gamma 1 for DNA synthesis inducing activity, cDNA fragments encoding Src homology (SH) and pleckstrin homology (PH) domains were subcloned into the bacterial expression plasmid pGEX-2TK, and the GST fusion proteins were purified. The complete PLC gamma l SH domain peptide was found to induce DNA synthesis after microinjection into growth arrested fibroblasts. Peptides containing a single SH3 domain or two SH2 domains induced a partial response that was restored to full activity if they were co-injected. The PH domain peptide did not induce DNA synthesis. Thus, both SH3 and SH2 activity combine to give maximum DNA synthesis induction, demonstrating that non-catalytic structural domains of PLC gamma 1 have pronounced effects on mitogenic signaling pathways.

3T3 Cells↗