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Biomedical subjects

M R Smith

Publications and source records attributed to M R Smith.

At least 19 recordsLinked to original sources
PubMed1992

Components of the protein kinase C pathway induce Ia expression after injection into macrophages.

Macrophages are activated by a variety of microbial and cytokine stimuli. One feature of activation is the induction of class II Ag (Ia) on the cell surface. To understand the intracellular events that occur during activation, we investigated various agents with intracellular activities, and examined their effects on the induction of Ia. We first noted that several agents that activate protein kinase C (PKC) induced Ia, and that several inhibitors of PKC inhibited Ia induction by IFN-gamma. To directly test whether PKC induced Ia, we microinjected normal peritoneal macrophages with this enzyme and other intracellular mediators, then examined Ia expression. We observed that injection of PKC itself, or of other intracellular proteins thought to participate in the PKC pathway (Ras or phospholipase C gamma) strongly induced Ia expression. The Ia-inducing activity of transforming Ras protein was blocked by kinase inhibitor treatment of cells, suggesting that Ras signal transduction requires kinase activity. On the other hand, components of the protein kinase A pathway (phospholipase A2 and protein kinase A itself) did not induce Ia. Thus, the PKC pathway can control expression of macrophage surface Ia, possibly by regulating the genes of the MHC, and may play many other roles in the activation of macrophages.

Alkaloids↗
PubMed1992

A human B-cell lymphoma line with a de novo multidrug resistance phenotype.

A human diffuse large cell lymphoma line (WSU-DLCL) expressing multidrug resistance (MDR) was established from a patient with primary chemotherapy-resistant disease. This cell line has the same phenotypic features as malignant cells from the patient. The established cell line has features of a mature B-cell neoplasm with no evidence for commitment to other lineages. WSU-DLCL grows in suspension forming relatively large clumps of cells with a doubling time of 20 hours. By light microscopic examination, the cells are very large with primitive lymphoid features, have a large amount of cytoplasm containing numerous vacuoles and an irregular outline. Immunophenotypic characterization by monoclonal antibodies and flow cytometric analysis showed a monoclonal IgM kappa B-cell phenotype with high expression of the multidrug-resistant P-glycoprotein compared with either normal peripheral blood lymphocytes or cells of the REH cell line. The cells were negative for T-cell and myeloid/monocyte antigens as well as Epstein-Barr virus nuclear antigen (EBNA). In addition, the cell line expressed high levels of MDR RNA. DNA histogram generated by flow cytometry indicated a DNA index of 1.83. Cytogenetic analysis confirmed hypertriploidy and showed complex chromosomal abnormalities including 14q+. This cell line should be a valuable tool to study the role of the MDR gene in the primary resistance of lymphomas to chemotherapy and to facilitate therapeutic investigations.

Adult↗
PubMed1992

Bone marrow necrosis in lymphoma studied by magnetic resonance imaging.

We used magnetic resonance imaging (MRI) to examine the extent of necrosis in the marrow space in three patients with lymphoma who had necrosis in bone marrow biopsies taken from the posterior iliac crest. Two of the patients had multiple focal lesions observed in the spine, pelvis, and femurs. These lesions had MRI characteristics of fluid-filled necrosis within marrow or lymphoma. The third patient exhibited a unique pattern of anatomically extensive marginated lesions otherwise typical of avascular necrosis of bone. These cases provide evidence that MRI can document the extent of necrosis within the marrow space and can differentiate two distinct patterns of necrosis.

Adult↗
PubMed1992

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.

Adult↗
PubMed1992

Characterization of a novel nuclear localization signal in the HTLV-I tax transactivator protein.

The Tax trans-activator protein of the type I human T-cell leukemia virus is expressed predominantly in the nuclei of cells. However, this viral trans-activator is distinguished from most other nuclear proteins by the absence of a short highly basic nuclear localization signal. Previous mutational analyses of the tax gene revealed that many of the missense mutations involving the amino terminus of Tax resulted in a predominantly cytoplasmic pattern of expression. We now report that the amino terminal 48 residues of Tax comprise a functional nuclear localization signal as demonstrated by the ability of this region to retarget expression of a large cytoplasmic protein to the nucleus.

Amino Acid Sequence↗
PubMed1992

Intraperitoneal P-32 is not an effective consolidation therapy after a negative second-look laparotomy for epithelial carcinoma of the ovary.

Thirty-four patients with epithelial carcinoma of the ovary were entered into a trial of adjuvant intraperitoneal P-32 following induction chemotherapy and a negative second-look laparotomy. The breakdown by initial Stage was Stage IC, 5; Stage II, 3; Stage III optimal, 22; and Stage III suboptimal, 4. Previous treatment consisted of 4-12 cycles (median 6) of cisplatin or carboplatin-based combination chemotherapy. Fifteen millicuries of P-32 were instilled via a Tenckhoff catheter placed at the time of second-look laparotomy. Because of a 22% incidence of bowel injury in the first 23 patients, the P-32 dose was reduced to 12 mCi in the last 11 patients. To date, there have been no bowel injuries at the lower dose. Eighteen of the 34 (53%) patients have relapsed with a median time to relapse of 20 months and a median follow-up for all patients of 31 months. There has been no difference in the relapse rate between a dose of 12 and 15 mCi. Intraperitoneal P-32 does not appear to reduce the relapse rate following a negative second-look laparotomy. The incidence of bowel injury is dose dependent and is higher than that seen in patients treated as an adjuvant following initial surgery without subsequent chemotherapy or second-look laparotomy.

Adult↗
PubMed1992

Emergence of myeloid stem cell line from T-lymphoid blastic phase of chronic myeloid leukemia in culture.

A new cell line designated JA-CML was derived from the peripheral blood of a patient with blastic phase CML. Sequential evolution of phenotypic and genetic markers was demonstrated during adaptation from primary to continuous culture in vitro. In the primary sample the majority of blast cells displayed the early T-cell markers, CD7, HLA-DR, and TdT, but were negative for the common ALL antigen (CALLA), CD4 and CD8. Simultaneously, unstimulated metaphase cells showed great karyotypic variation with a range of 43-46 chromosomes per cell. Clonal changes included the Ph chromosome t(9;22), loss of the Y and gain of several altered chromosomes. The cells grew slowly in suspension during the first 10 weeks of culture. During that time, cells still expressed the CD7 and HLA-DR antigens. Karyotypic analysis at ten weeks showed a pattern of 46,X,-Y,t(9;22),+8 in more than 90% of metaphases with disappearance of all other abnormal chromosomes noted in the original sample. A tetraploid subline exhibiting duplication of most chromosomes, including the Ph, comprised the remaining metaphases. Upon further cultivation in vitro, the cells transformed spontaneously over a period of several weeks, from T-lymphoid into myeloid cells. Expression of CD7 was lost, but reactivities with monoclonal antibodies to CD34, CD33 and CD13 were newly acquired. The karyotype was hypertriploid and all cells carried two copies of t(9;22) and lacked normal copies of No. 9 or Y. The cells have since maintained stable cytogenetic and phenotypic profiles. Molecular rearrangement of the breakpoint cluster region was identified in the primary blasts and the established line and T-cell receptor gene rearrangements were not found. These observations suggest that the leukemic blast arose from primitive stem cells, not irreversibly committed T cells, and that these stem cells retained the capacity to differentiate along the myeloid pathway.

Antigens, CD↗
PubMed1992

Regulation of intracellular actin polymerization by prenylated cellular proteins.

Posttranslational modification by covalent attachment of polyisoprene intermediates to a carboxyterminal CAAX-box motif is required for the biologic function of proteins such as p21ras, the supergene family of ras-related proteins, nuclear lamins, and subunits of heterotrimeric G-proteins. Cells grown in the presence of lovastatin, which inhibits HMG-CoA reductase and prevents synthesis of intermediates required for protein prenylation, develop a round, refractile morphology. Our data indicate that this is due to the selective loss of actin cables without gross changes in the microtubular lattice or intermediate filament structure. Microinjection of a competitive peptide inhibitor of protein prenyltransferases into the cytoplasm of cells induces an identical change in morphology with loss of actin cables. Mevalonate (MVA) reverses the lovastatin-induced morphologic change by inducing a rapid repolymerization of actin cables with coincident reversion to the flat morphology. Furthermore, microinjection of farnesyl-pyrophosphate or geranylgeranyl-pyrophosphate into lovastatin-treated cells also results in rapid morphologic reversion. The morphologic reversion induced by MVA requires the presence of serum, and is independent of extracellular calcium. The addition of cycloheximide to the growth medium prevents lovastatin-induced loss of actin cables, and causes morphologic reversion of lovastatin-treated cells by a mechanism that is independent of MVA. A1F4- induces morphologic reversion in a manner indistinguishable from MVA. These data indicate that prenylated protein(s) play a critical role in regulating the state of intracellular actin, and that GGPP can rescue the lovastatin-induced morphologic phenotype in the absence of upstream intermediates of cholesterol biosynthesis. We have begun to dissect the signaling events that mediate this pathway.

Actin Cytoskeleton↗
PubMed1992

Ras mediates translation initiation factor 4E-induced malignant transformation.

Translation initiation factor eIF-4E binds to the eukaryotic mRNA 5' cap structure (m7 GpppN, where N is any nucleotide). eIF-4E is a limiting factor in translation and plays a key role in regulation of translation. We have shown previously that overexpression of eIF-4E in rodent fibroblasts results in tumorigenic transformation. eIF-4E also exhibits mitogenic activity when microinjected into serum-starved NIH-3T3 cells. To understand the mechanisms by which eIF-4E exerts its mitogenic property, we examined the involvement of the Ras signaling pathway in this activity. Here, we report that Ras is activated in eIF-4E-overexpressing cells, as the proportion of GTP-bound Ras is increased. Overexpression of the negative effector of cellular Ras, GTPase activating protein, causes reversion of the transformed phenotype. Furthermore, we show that neutralizing antibodies to Ras, or a dominant-negative mutant of Ras, inhibit the mitogenic activity of eIF-4E. We conclude that eIF-4E exerts its mitogenic and oncogenic activities by the activation of Ras.

3T3 Cells↗
PubMed1992

EGF receptor activity and mitogenic response of Balb/3T3 cells expressing Ras and Myc oncogenes. EGF receptor activity in oncogene transformed cells.

EGF receptors are found on the surface of most cells, usually with high and low binding affinities. To investigate functional relationships between EGF (EGF-like growth factors) and oncogenes we have characterized the expression of the epidermal growth factor receptor (EGFr) in H-Ras, v-Myc, and H-Ras-v-Myc transformed Balb/3T3 cells. H-Ras cells show a marked decrease in the number of EGFr molecules per cell compared to parental cells. v-Myc and H-Ras-v-Myc transformed cells express an intermediate level of receptors. The majority of the EGF receptors on the parental and oncogene transformed cells bind EGF with low affinity and this low affinity receptor is down-regulated by oncogene transformation. v-Myc expression, in the H-Ras-v-Myc transformed cells, abrogates the receptor down-regulation seen with H-Ras transformation. The mechanism of abrogation is not a result of a change in the p21-Ras concentration in the H-Ras-v-Myc transformed cells. In addition, the mitogenic response to EGF was examined. H-Ras and H-Ras-v-Myc transformed cells do not respond to EGF mitogenically. In contrast, EGF stimulates DNA synthesis in parental cells and v-Myc transfected cells; this result suggests that growth promoting signals from the EGF receptor may not be required in H-Ras transformed cells.

3T3 Cells↗
PubMed1991

Evidence for clonal disease by magnetic resonance imaging in patients with hypoplastic marrow disorders.

Some patients with hypoplastic marrow disorders, including aplastic anemia (AA), are at risk for clonal evolution to myelodysplastic syndromes (MDS) and leukemia. Magnetic resonance imaging (MRI) of marrow of the spine, pelvis, and femurs was performed in 24 patients with hypoplastic marrow disorders. In 12 patients (three AA, nine MDS) MRI was compatible with the clinical and biopsy diagnoses and served to define the spectrum of marrow patterns in these disorders. In eight patients with hypocellular marrow biopsies and a clinical diagnosis of AA, MRI showed an unexpected inhomogeneous or diffuse cellular pattern. Concurrent or subsequent marrow or cytogenetic studies have led to diagnoses of hypoplastic MDS in seven of these patients. In four patients with prolonged hypoplasia after bone marrow transplantation for lymphoma, a speckled pattern superimposed on a fatty background appeared in serial MRI studies. One case evolved to AML, two developed megaloblastic foci, and one remains hypoplastic at 19 months. This study suggests that MRI is able to detect early clonal disease in patients with AA, and can distinguish AA from hypoplastic MDS.

Adult↗
PubMed1991

IL-1 induces ornithine decarboxylase in normal T lymphocytes.

IL-1 alpha regulation of ornithine decarboxylase (ODC) was examined in T cells because IL-1 is a costimulus for T cell proliferation and ODC is a critical enzyme in the metabolic events associated with cellular proliferation. In the present study, we demonstrate that IL-1 alpha induces ODC mRNA and ODC enzyme activity in T cells. Unlike many IL-1 actions on T cells, this did not require a costimulus from the TCR, IL-1 alone being sufficient to induce ODC. The mechanism of IL-1 induction of ODC probably operates at several levels, including transcription, mRNA stability, and translation. Previous studies have shown that IL-1 prepares T cells for replication by increasing the production of c-jun, c-fos, c-myc, growth factors, growth factor receptors, and the response to growth factors. From the present study, ODC induction can be added to the list of IL-1-induced replicative machinery in T cells.

Animals↗
PubMed1991

Successful management of Ph chromosome chronic myelogenous leukemia with leukapheresis during pregnancy.

We report the successful management of a 25 year-old woman diagnosed in the second trimester of her pregnancy with chronic myelogenous leukemia (CML). She was treated with leukapheresis until her delivery at 38 weeks of gestation. The procedure was without significant adverse effects on the patient or the fetus. Following induced vaginal delivery, the patient underwent allogeneic bone marrow transplantation from her HLA-matched brother and is presently disease free 13 months following her transplant.

Adult↗
PubMed1991

A quantitative comparison of the TERA modeling and DFT magnetic resonance image reconstruction techniques.

The resolution of magnetic resonance images reconstructed using the discrete Fourier transform (DFT) algorithm is limited by the effective window generated by the finite data length. The transient error reconstruction approach (TERA) is an alternative reconstruction method based on autoregressive moving average (ARMA) modeling techniques. Quantitative measurements comparing the truncation artifacts present during DFT and TERA image reconstruction show that the modeling method substantially reduces these artifacts on "full" (256 X 256), "truncated" (256 X 192), and "severely truncated" (256 X 128) data sets without introducing the global amplitude distortion found in other modeling techniques. Two global measures for determining the success of modeling are suggested. Problem areas for one-dimensional modeling are examined and reasons for considering two-dimensional modeling discussed. Analysis of both medical and phantom data reconstructions are presented.

Algorithms↗
PubMed1991

Degradation of 3-chloro-2-methylpropionic acid by Xanthobacter sp. CIMW 99.

Both stereoisomers of 3-chloro-2-methylpropionic acid (CMPA) and its methyl esters (MeCMPA) serve as growth substrates for a bacterial isolate (Xanthobacter sp. CIMW 99) when supplied as sole source of carbon and energy. Biodegradation of DL-CMPA and DL-MeCMPA was shown to be via a common pathway; an initial, constitutive, esterase converted the methyl ester to the corresponding carboxylic acid. Further metabolism required the activation of CMPA involving a CoA-, ATP-, Mg(2+)-dependent chloroacyl-CoA synthetase. Most noteworthy, it was the product of this reaction (3-chloro-2-methylpropionyl-CoA) that underwent hydrolytic dehalogenation to give 3-hydroxy-2-methylpropionyl-CoA (3-hydroxyisobutyryl-CoA). Further biodegradation proceeded by the action of a dehydrogenase on the CoA derivative to give methylmalonate-CoA-semialdehyde. Cells of CIMW 99 also contained a stable, constitutive, highly active 3-hydroxyisobutyrate dehydrogenase that was specific for the L(+) isomer. However, evidence is presented suggesting that this enzyme was not involved in the catabolism of the chlorinated substrates.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Human T-cell leukemia virus type I Tax induces expression of the Rel-related family of kappa B enhancer-binding proteins: evidence for a pretranslational component of regulation.

The Tax protein of the human T-cell leukemia virus type I (HTLV-I) serves as a potent transcriptional activator of its own long terminal repeat as well as select cellular genes, including interleukin-2 and the alpha subunit of the interleukin-2 receptor. Tax activation of these two growth-related genes appears to involve the induced nuclear expression of DNA-binding proteins that specifically engage related kappa B enhancer elements present in the 5' regulatory regions of these genes. In human T cells, kappa B enhancer-binding activity has been discerned as an unexpectedly large family of UV cross-linked nucleoprotein adducts, termed p50, p55, p75, and p85. The protein components of each of these DNA-protein adducts have been shown to share structural similarity with the v-rel oncogene product. The p55 adduct is composed of the 50-kDa subunit of NF-kappa B derived from a 105-kDa precursor polypeptide, while the p50 adduct contains a smaller protein that is closely related to NF-kappa B p50. The p75 adduct contains the 65-kDa subunit of NF-kappa B, while the p85 adduct is composed of the human c-rel proto-oncogene product. We now demonstrate that HTLV-I Tax, in the absence of other viral pX gene products, is capable of inducing the nuclear expression of all four of these kappa B-binding proteins in human T cells, with most marked effects involving c-Rel and NF-kappa B p65. Tax induction of the nuclear expression of c-Rel and NF-kappa B p50 is regulated, at least in part, at a pretranslational level involving increases in c-rel and NF-kappa B p105 mRNA expression. To study the pattern of expression of these kappa B-specific proteins in cells infected with the whole HTLV-I, seven cloned HTLV-I-infected T-cell lines were established from the peripheral blood of patients with adult T-cell leukemia. Of note, only three of these seven cell lines produced Tax, and c-rel mRNA and nuclear protein expression was confined to these three cell lines. In contrast, NF-kappa B p50 and NF-kappa B p65 were constitutively expressed in the nuclei of all seven of the HTLV-I-infected cell lines, even in the absence of detectable Tax or other viral gene expression. These findings raise the possibility of an alternate, Tax-independent pathway for the induced nuclear expression of NF-kappa B p50 and NF-kappa B p65 following HTLV-I infection.

Blotting, Northern↗