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Biomedical subjects

M B Prystowsky

Publications and source records attributed to M B Prystowsky.

At least 55 records · Page 3Linked to original sources
PubMed1992

Expression of the T-lymphocyte activation gene, F5, by mature neurons.

F5 was first identified as an mRNA expressed by activated but not resting T-lymphocytes. Subsequent studies suggested that it also is selectively expressed by mature neurons. Although the F5 protein coding sequence is highly conserved, the function of the F5-encoded protein is unknown. The present studies were undertaken to define the anatomic distribution, cellular specificity, and developmental pattern of F5 mRNA expression in the mouse nervous system, addressing specifically the question of whether the expression pattern of F5 corresponds to that of known ligand-receptor or signal-transduction systems. The use of a nonradioactive in situ hybridization method and paraffin-embedded sections provided excellent morphological preservation and a high degree of cellular resolution. F5 mRNA was detected in the central nervous system, peripheral nervous system, and retina in cells having the location and morphological features of neurons. Combined in situ hybridization histochemistry for F5 mRNA and immunofluorescence staining for cell-specific markers confirmed that neurons expressed F5 mRNA but astrocytes did not. The neuronal expression of F5 mRNA had two interesting features. First, the level of expression appeared to correlate directly with the size of the neuronal perikarya, the length of the axonal projection, or the extent of dendritic arborization. Second, F5 mRNA appeared late in post-natal development. These observations are of interest because of preliminary data suggesting that F5 may function as a substrate for protein kinase C, which demonstrates a similar expression pattern in the nervous system.

Animals↗
PubMed1992

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.

Amino Acid Sequence↗
PubMed1992

Requirement for prolactin during cell cycle regulated gene expression in cloned T-lymphocytes.

The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated lymphocyte proliferation. Since the sequential expression of cell cycle regulated genes occurs during this process, we examined the contribution of IL2 and PRL to specific RNA accumulation. Stimulation of the cloned T cell line L2 with IL2 and PRL induced the sequential expression of interferon regulatory factor-1, c-myc, proliferating cell nuclear antigen, thymidine kinase, cyclin B, and histone H3. Stimulation of L2 cells with PRL alone, however, induced only the expression of interferon regulatory factor-1. Depletion of PRL, through the use of an anti-PRL antiserum, inhibited IL2 driven proliferation and the expression of cyclin B and histone H3. These results demonstrate that PRL may regulate T cell proliferation by enhancing the expression of some genes necessary for entry into S-phase.

Animals↗
PubMed1991

Surface marker analysis and karyotype distinguish acute biphenotypic leukemia from acute myelogenous leukemia expressing terminal deoxynucleotidyl transferase.

Surface phenotyping by flow cytometry and cytochemical study were used to identify 15 adult patients with acute leukemia displaying ambiguous phenotypes. Differences were found in the blast cell karyotype and immunoglobulin gene rearrangements of terminal deoxynucleotidyl transferase (TdT)-positive acute myelogenous leukemia (AML) and biphenotypic leukemia expressing B lymphoid and myeloid markers. The karyotypic abnormalities, t(9;22) and t(4;11), were noticed in acute biphenotypic leukemia, and were consistently associated with rearrangement at the immunoglobulin locus. Furthermore, coexpression of CD19/CD20 and either myeloperoxidase or myeloid surface markers were predictive of finding the t(9;22) or t(4;11) karyotype. Patients with TdT-positive AML, on the other hand, were less likely to show rearrangement at the immunoglobulin locus, and did not have the t(9;22) or t(4;11). Instead, a variety of nonrandom karyotypic abnormalities were seen, including trisomy 13. Unlike common AML, the majority of TdT-positive cases demonstrated an abnormal karyotype with duplications and/or deletions present in all cases. In no instance was trisomy 8, t(8;21), t(15;17), or any other isolated translocation identified. The authors therefore suggest that immunophenotyping, when combined with cytochemical analysis of TdT and myeloperoxidase or Sudan black B, may aid in the characterization of subgroups of atypical acute leukemia, such that alternate approaches to therapy can be evaluated.

Antigens, Neoplasm↗
PubMed1991

Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development.

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.

Animals↗
PubMed1991

Requirement of nuclear prolactin for interleukin-2--stimulated proliferation of T lymphocytes.

Prolactin (PRL) is necessary for the proliferation of cloned T lymphocytes in response to interleukin-2 (IL-2). Translocation of PRL into the nucleus occurs during IL-2--stimulated mitogenesis. Therefore, the function of intranuclear PRL in T cell proliferation was tested. Eukaryotic expression vectors were prepared to express wild-type PRL [PRL(WT)], PRL that lacks the signal sequence for translocation into the endoplasmic reticulum [PRL(ER-)], and chimeric PRL in which the signal peptide was replaced with the sequence that directs the nuclear translocation of the SV40 large T antigen [PRL(NT+)]. Expression of these constructs in a T cell line (Nb2) responsive to PRL and IL-2 resulted in localization of PRL in the extracellular milieu, cytoplasm, or nucleus, respectively. Stimulation with IL-2 alone resulted in a five- to tenfold increase in the incorporation of [3H]thymidine by cells expressing PRL(NT+) or PRL(WT) as compared to PRL(ER-) or the parental Nb2 cells. Only the PRL(NT+) clone proliferated continuously with IL-2 stimulation in the presence of antiserum to PRL. These results demonstrate that nuclear PRL is necessary for IL-2--stimulated proliferation and suggest that a peptide hormone can function in the nucleus without binding to its cell surface receptor.

Amino Acid Sequence↗
PubMed1991

The kinetics of vimentin RNA and protein expression in interleukin 2-stimulated T lymphocytes.

In this paper, we examine the regulation of vimentin expression during interleukin 2-induced proliferation of the cloned helper T cell line, L2. We observe a 10-20-fold increase in steady-state vimentin RNA that is accompanied by a 14-fold increase in the rate of vimentin protein synthesis. This indicates that translation is occurring, and thus that vimentin expression is regulated, at least in part, at the level of transcription and/or RNA stability, rather than at the level of protein synthesis. In contrast to the increases in steady-state vimentin RNA and the rate of protein synthesis, steady-state vimentin protein levels increase maximally only 1.3-3-fold in proliferating L2 cells. The rate of vimentin protein turnover remains relatively constant throughout L2 cell activation, active proliferation, and return to quiescence, indicating that an increased rate of turnover does not account for the minimal increase in vimentin content. These observations are consistent with a transient increase in vimentin protein synthesis necessary to support L2 cell growth and/or vimentin network reorganization. Because cell division occurs before an equilibrium state is reached, the maximal increase in steady-state vimentin protein content is never attained.

Blood Proteins↗
PubMed1991

Single proline substitutions in predicted alpha-helices of murine granulocyte-macrophage colony-stimulating factor result in a loss in bioactivity and altered glycosylation.

Contributions of alpha-helices to biological activity in murine granulocyte-macrophage colony-stimulating factor were analyzed using site-directed mutagenesis and protein expression in COS-1 cells. A series of single proline substitutions were made for residues within the four predicted alpha-helices as a means of disrupting local helical secondary structure. Mutations in three of the four helices resulted in marked reductions in bioactivity. Five mutants E21P, L56P, E60P, L63P, and L107P showed 10(2)-10(4)-fold reduction in bioactivity as well as hyperglycosylation. The same Pro substitutions made on non-N-glycosylated molecules had a similar loss in bioactivity implying that a Pro-induced structural change and not hyperglycosylation was responsible for the major decrease in bioactivity. Additional amino acid substitutions at these residues which conserved charge or hydrophobicity, or replaced the original residue with an Ala, verified that conformational changes in the protein structure were specifically due to steric constraints imposed by the Pro residue rather than loss of important side chain functions.

Amino Acid Sequence↗
PubMed1991

A neutralizing monoclonal antibody binds to an epitope near the amino terminus of murine granulocyte-macrophage colony-stimulating factor.

A rat anti-murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) monoclonal antibody, A2, that neutralizes bioactivity in vitro was isolated. The binding epitope recognized by this antibody was identified using human-murine hybrid GM-CSF proteins. A2 was unable to immunoprecipitate a hybrid (hm7) protein containing the human GM-CSF sequence for the first 11 amino terminal amino acids, and the mGM-CSF sequence for amino acids 12-124. In contrast, A2 did recognize a hybrid which substitutes human GM-CSF amino acids 23-36 in the murine sequence. These data suggest that this neutralizing antibody recognizes an epitope at the amino terminus of mGM-CSF. Because hm7 did maintain in vitro bioactivity, it is probable that the epitope recognized by the neutralizing antibody is not itself part of the receptor-binding domain of mGM-CSF; rather, it is likely that neutralization occurs as a result of antibody binding near the receptor-binding site, with steric inhibition of mGM-CSF binding to its receptor. Interestingly, monoclonal antibody A2 does not recognize mGM-CSF glycosylation species corresponding to predicted maximal O-glycosylation variants. The presence of O-glycosylation sites within the antibody-binding epitope was confirmed using site-directed mutagenesis. Potential O-glycosylation sites in native mGM-CSF were removed by introducing conservative amino acid substitutions, and expected molecular weight reductions were obtained. These findings are consistent with previous reports that suggest the importance of the integrity of residues near the amino terminus to GM-CSF bioactivity.

Amino Acid Sequence↗
PubMed1991

Novel expression pattern of a new member of the MIP-1 family of cytokine-like genes.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) specifically induces the growth of myeloid progenitors and their maturation into neutrophils and macrophages. We have identified a series of previously uncharacterized hematopoietic-specific mRNAs that are expressed in myelopoietic mouse bone marrow cultures stimulated by GM-CSF. One of these messages, C10, encodes a new member of the family of cytokine-like genes related to macrophage inflammatory protein-1 (MIP-1). Members of this family are all induced by one or more stimuli related to inflammation, wound repair, or immune response. In contrast, C10 mRNA showed little or no accumulation in response to such activating agents and was greatly reduced on activation of a T-cell line. On the other hand, C10 mRNA, unlike MIP-1, was acutely stimulated during the first day of bone marrow culture in GM-CSF, and it was also strongly elevated during the induction of neutrophilic differentiation of 32D cl3 cells by granulocyte colony-stimulating factor. The implications of this unusual expression pattern are discussed.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of murine PCNA: interspecies comparison of the cDNA and the 5' flanking region of the gene.

Proliferating cell nuclear antigen (PCNA) RNA levels are regulated by transcription as well as changes in stability, in growing cells. We have cloned the murine PCNA cDNA and a fragment of the murine PCNA gene flanking the transcription initiation site. Comparison of the murine deduced amino acid sequence with the PCNA sequence from rat, human, Drosophila, Saccharomyces cerevisiae, and higher plants, reveals extensive homology between species. The homology is likely to be related to the fundamental role of PCNA as an auxiliary protein for DNA replication. Consensus sequences for transcriptional regulatory factors identified within 520 bp 5' of the cap site of the murine PCNA gene include: an inverted CCAAT site, an enhancer core element (EBP-1), three cAMP-response elements (CRE-BP), one AP-2 site, three Sp1 sites, and two octamer sequences. The first 20 bp of the transcriptional unit are homologous to an initiator element, which may direct transcription from RNA polymerase II in the absence of a TATAA box. The consensus elements in the murine PCNA gene are similar in sequence and/or location to elements identified in the genes for human, Drosophilia, and yeast PCNA.

Amino Acid Sequence↗
PubMed1991

Combined therapy for Sézary syndrome with extracorporeal photochemotherapy and low-dose interferon alfa therapy. Clinical, molecular, and immunologic observations.

Extracorporeal photochemotherapy is a well-tolerated treatment of palliative benefit for patients with the Sézary syndrome, but up to 25% of patients do not respond to this form of therapy. Interferon alfa is a glycoprotein with immunomodulatory properties that is also an active agent in the treatment of Sézary syndrome; however, dose-related toxic effects often limit its long-term use. A patient with rapidly advancing Sézary syndrome was treated with a combination of extracorporeal photochemotherapy and low doses of subcutaneous interferon alfa-2b. In addition to the almost complete clearing of his skin lesions and the marked diminution of his extensive lymphadenopathy after 16 weeks of combined therapy, disappearance of the malignant T-cell clone from the peripheral blood was documented by Southern blot analysis. Normalization of the peripheral blood lymphocyte phenotypic profile and an increase in natural killer cell activity were also observed. The excellent clinical response to combined therapy has been sustained, without adverse effects, during 18 months of treatment and 22 months of continued follow-up. The long-term disappearance of the malignant clone from the blood of a patient with Sézary syndrome in response to therapy has not been previously described and thus represents a novel observation. Extracorporeal photochemotherapy and low-dose interferon alfa are well-tolerated therapies that, when combined, may have additive efficacy in the treatment of Sézary syndrome. These results suggest that further exploration of this therapeutic combination is warranted.

Combined Modality Therapy↗
PubMed1990

Interleukin 2-induced expression of proliferating cell nuclear antigen is regulated by transcriptional and post-transcriptional mechanisms.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein required for leading strand DNA synthesis by polymerase delta. Steady-state levels of PCNA RNA increase during interleukin 2 stimulated G1 activation of cloned T cells (L2 cells) and correlate with expression of the PCNA protein. We demonstrate that the initial (G1) rapid rise in PCNA RNA levels is predominantly related to increased transcription. The slower rate of increase in PCNA RNA at the initial G1-S transition is related to transcription as well as increased stability of the PCNA message and partially requires protein synthesis. These data provide a foundation for further investigation of the mechanisms of increased stability and transcription of PCNA.

Animals↗
PubMed1990

cDNA cloning and characterization of interleukin 2-induced genes in a cloned T helper lymphocyte.

Clonal expansion of antigen-specific lymphocytes is an important aspect of the immune response. Interleukin 2 (IL2) is largely responsible for the amplification of antigen-specific T cells. In this study, the changes in gene expression accompanying interleukin 2 stimulation of T cells are examined, using a cloned T helper lymphocyte line as a model system. To isolate cDNA clones of IL2-induced genes, a cDNA library was screened by differential hybridization. Twenty-one different cDNA clones were isolated by this method, comprising six glycolytic enzymes, vimentin, alpha-tubulin, beta-actin, gamma-actin, ERp99, elongation factor 2, ribosomal phosphoprotein P1, the DNA-binding protein dbpB/YB-1, as well as seven clones which do not correspond to any previously described sequences. These clones are used to study the time course of expression and the sensitivity to cycloheximide inhibition of IL2-induced mRNAs. In addition, the tissue specificity of the unidentified mRNAs is examined, and two of these are shown to be expressed at high levels in normal mouse brain, with much lower or undetectable levels in the other tissues tested. These cDNA clones will be useful in future studies to determine the molecular basis of IL2-induced gene expression.

Animals↗
PubMed1990

Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned T-helper lymphocyte.

A cDNA library was constructed using mRNA from interleukin 2 (IL2)-stimulated cloned murine T lymphocytes to isolate cDNA clones of mRNAs that were induced by IL2 and present at maximal levels in late G1/early S phase of the cell cycle. When the library was screened by differential hybridization, over half of the clones isolated were found to cross-hybridize, indicating that there was a predominant IL2-induced mRNA in these cells. This cDNA was identified as encoding murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). The in vitro translation product of this cDNA was a 36-kDa protein using both hybridization-selected RNA and in vitro transcribed RNA. We estimate that GAPDH mRNA comprises approx. 0.7% of total mRNA in the cloned T cells in late G1. GAPDH mRNA is induced two- to fivefold over resting levels upon IL2 stimulation, due in part to an increased rate of transcription. GAPDH enzymatic activity is induced approx. sevenfold over resting levels. The induction of GAPDH mRNA is inhibited only slightly by CHX under conditions in which cell proliferation is inhibited. In addition, the induction of GAPDH is directly due to the effect of IL2, and not in conjunction with any serum components, since IL2 will induce GAPDH mRNA under serum-free conditions. Finally, when genomic DNA is probed with a full-length GAPDH cDNA, a complex pattern of bands is observed, whereas if a 5' end probe is used, a much simpler pattern is obtained, indicating that many of the GAPDH pseudogenes in the murine genome lack 5' sequence information.

Amino Acid Sequence↗
PubMed1990

Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation.

A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro. Northern blot analysis using poly(A)+ RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species with N-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length GM-CSF from tunicamycin-treated cells. Since very recent scanning-deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GM-CSF.

Amino Acid Sequence↗
PubMed1990

Regulation of interleukin 2-driven T-lymphocyte proliferation by prolactin.

The requirement for prolactin in interleukin 2-driven T-cell proliferation was evaluated. Addition of an anti-prolactin antiserum resulted in the specific inhibition of T-cell proliferation in a time- and dose-dependent manner. Synthesis of prolactin and its mRNA, however, did not occur during interleukin 2 stimulation. Instead, previously internalized prolactin, presumably from fetal bovine serum, appears to serve as the source of prolactin under serum-free conditions. A 7-fold increase in a prolactin receptor occurred as a function of cell cycle progression; accumulation of a 1.6-kilobase prolactin receptor mRNA increased approximately 2-fold. Interleukin 2 stimulation induced the translocation of prolactin into the nucleus and prolactin receptor to the nuclear periphery. These data indicate that extracellular prolactin is requisite for T-cell proliferation and suggest that the effects of prolactin are exerted in the nucleus.

Animals↗