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M S Rao

Publications and source records attributed to M S Rao.

At least 19 recordsLinked to original sources
PubMed1998

Implication of hydrogen peroxide generation and apoptosis in the neoplastic transformation of mouse fibroblasts overexpressing peroxisomal fatty acyl-CoA oxidase.

Receptor-mediated overexpression of H2O2-generating peroxisomal fatty acyl-CoA oxidase (AOX) has been implicated in peroxisome proliferator-induced hepatocarcinogenesis. To investigate the role of rat AOX generated H2O2 in transformation, we overexpressed this enzyme in a non-tumorigenic mouse fibroblast cell line (LM tk-) under control of mouse urinary protein promoter. The clones overexpressing rat peroxisomal AOX, when exposed to a fatty acid substrate (100 microM linoleic acid) for 6 to 96 h, demonstrated > 10-fold increase of intracellular H2O2. This increase in H2O2 concentration was associated with increased apoptosis as evidenced by DNA fragmentation, in situ terminal deoxynucleotide transferase dUTP nick end-labeling (TUNEL). These cell lines stably expressing AOX formed colonies in soft agar in proportion to the duration (1-7 weeks) of exposure to a fatty acid substrate (100 microM linoleic acid, erucic acid or nervonic acid) and these transformants developed into fibrosarcomas when injected in athymic nude mice. These results suggest that H2O2 generated by AOX overexpression in immortalized fibroblasts leads to apoptosis, and the extent and duration of H2O2 and possibly other DNA damaging reactive oxygen species generated by the overexpression of peroxisomal AOX can influence apoptosis and neoplastic transformation.

Acyl-CoA Oxidase↗
PubMed1997

New isoforms of the chick glutamate receptor subunit GluR4: molecular cloning, regional expression and developmental analysis.

To identify chick GluR4 isoforms, we used PCR to amplify a C-terminal region that is the site of alternative splicing in rat. We report here the cloning of three novel chick GluR4 isoforms. GluR4c has a 113-bp insert in the C-terminus, is expressed in flip and flop isoforms, is most strongly expressed in the cerebellum, midbrain and forebrain, and appears from embryonic day (E) 2.5 through at least post-hatching day (P) 2, with a peak of expression at E17. GluR4d has a 184-bp segment inserted at the 4c splice site, occurs as flip and flop isoforms, is expressed most strongly in cerebellum, hindbrain and forebrain, and is present from E11 through P2, with peak expression at E17. GluR4s is a shortened form that lacks the nominal 4th transmembrane and flip/flop domains and shares a common C-terminal region with GluR4. GluR4s is expressed most strongly in the hindbrain and cerebellum and its expression increases from E11 through P2. Experiments on purified cerebellar cells show that glia express GluR4c and GluR4d at combined levels nearly twice that of GluR4 and that flip isoforms predominate. In contrast, granule cells express GluR4c and GluR4d at a level comparable to GluR4 and express GluR4s at a level less than half that in cerebellar glia. Thus, the independence of alternative splicing at the flip/flop and C-terminal splice sites allows seven alternatively spliced forms of GluR4 to exist in chick CNS. This structural diversity increases the potential for functional diversity in neuronal and glial GluRs incorporating GluR4.

Alternative Splicing↗
PubMed1997

Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor.

In an attempt to identify cofactors that could possibly influence the transcriptional activity of peroxisome proliferator-activated receptors (PPARs), we used a yeast two-hybrid system with Gal4-PPARgamma as bait to screen a mouse liver cDNA library and have identified steroid receptor coactivator-1 (SRC-1) as a PPAR transcriptional coactivator. We now report the isolation of a cDNA encoding a 165-kDa PPARgamma-binding protein, designated PBP which also serves as a coactivator. PBP also binds to PPARalpha, RARalpha, RXR, and TRbeta1, and this binding is increased in the presence of specific ligands. Deletion of the last 12 amino acids from the carboxyl terminus of PPARgamma results in the abolition of interaction between PBP and PPARgamma. PBP modestly increased the transcriptional activity of PPARgamma, and a truncated form of PBP (amino acids 487-735) acted as a dominant-negative repressor, suggesting that PBP is a genuine coactivator for PPAR. In addition, PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. In situ hybridization and Northern analysis showed that PBP is expressed in many tissues of adult mice, including the germinal epithelium of testis, where it appeared most abundant, and during ontogeny, suggesting a possible role for this cofactor in cellular proliferation and differentiation.

Amino Acid Sequence↗
PubMed1997

Glial-restricted precursors are derived from multipotent neuroepithelial stem cells.

Neuroepithelial cells in the developing ventricular zone differentiate into neurons, astrocytes, and oligodendrocytes. It is not known, however, whether this differentiation occurs in a single step or is a pathway utilizing intermediate more restricted precursor cells. To characterize the generation of glial cells from multipotent stem cells we have cultured neuroepithelial (NEP) cells from E10.5 rat embryos. Cultured NEP cells do not express any glial differentiation markers when grown on fibronectin/laminin under nondifferentiation conditions. NEP cells, however, differentiate into A2B5 immunoreactive cells which can subsequently give rise to oligodendrocytes and astrocytes. Clonal analysis of NEP cells demonstrates that the A2B5 immunoreactive cells arise in clones that contain neurons and astrocytes, indicating that A2B5(+) cells arise from multipotent NEP precursor cells. A2B5(+) cells, maintained as undifferentiated cells over multiple passages, can subsequently give rise to both oligodendrocytes and astrocytes. A2B5(+) cells, however, do not generate neurons. Thus A2B5(+) cells represent a restricted progenitor cell population that differentiates from a multipotent NEP cell. Based on our results we propose that differentiation of the multipotential NEP cells to terminally differentiated glial cells occurs via intermediate restricted precursors.

Animals↗
PubMed1997

Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.

To isolate mouse neural crest stem cells, we have generated a rat monoclonal antibody to murine neurotrophin receptor (p75). We have immortalized p75+ murine neural crest cells by expression of v-myc, and have isolated several clonal cell lines. These lines can be maintained in an undifferentiated state, or induced to differentiate by changing the culture conditions. One of these cell lines, MONC-1, is capable of generating peripheral neurons, glia, and melanocytic cells. Importantly, most individual MONC-1 cells are multipotent when analyzed at clonal density. The neurons that differentiate under standard conditions have an autonomic-like phenotype, but under different conditions can express markers of other peripheral neuronal lineages. These lines therefore exhibit a similar differentiation potential as their normal counterparts. Furthermore, they can be genetically modified or generated from mice of different genetic backgrounds, providing a useful tool for molecular studies of neural crest development.

Animals↗
PubMed1997

Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterization, and clonal analysis.

Adherent cultures of E10.5 rat neuroepithelial cells (NEP cells) from the caudal neural tube require FGF (fibroblast growth factor) and CEE (chick embryo extract) to proliferate and maintain an undifferentiated phenotype in culture. Epidermal growth factor (EGF) does not support E10.5 NEP cells in adherent culture and NEP cells do not form EGF-dependent neurospheres. NEP cells, however, can be grown as FGF-dependent neurospheres. NEP cells express nestin and lack all lineage-specific markers for neuronal and glial sublineages, retain their pleuripotent character over multiple passages, and can differentiate into neurons, astrocytes, and oligodendrocytes when plated on laminin in the absence of CEE. In clonal culture, NEP cells undergo self-renewal and generate colonies that vary in size from single cells to several thousand cells. With the exception of a few single-cell clones, all other NEP-derived clones contain more than one identified phenotype, with over 40% of the colonies containing A2B5, beta-111 tubulin, and GFAP-immunoreactive cells. Thus, NEP cells are multipotent and capable of generating multiple neural derivatives. NEP cells also differentiate into motoneurons immunoreactive for choline acetyl transferase (ChAT) and the low-affinity neurotrophin receptor (p75) in both mass and clonal culture. Double labeling of clones for ChAT and glial, neuronal, or oligodendrocytic lineage markers shows that motoneurons always arose in mixed cultures with other differentiated cells. Thus, NEP cells represent a common progenitor for motoneurons and other spinal cord cells. The relationship of NEP cells with other neural stem cells is discussed.

Animals↗
PubMed1997

alpha 1-antitrypsin deficiency-associated panniculitis: resolution with intravenous alpha 1-antitrypsin administration and liver transplantation.

Panniculitis is a rare complication of alpha 1-antitrypsin (A1AT) deficiency that is characterized by acute inflammatory infiltrate and fat necrosis. Different treatment strategies are used to provide symptomatic relief. Here we describe two patients with homozygous A1AT deficiency who developed panniculitis and were successfully treated with A1AT replacement. The patient who received a liver transplant experienced complete resolution of the skin lesions. The patient who received A1AT intravenously showed complete response, but the skin lesions recurred when the levels of A1AT fell below 50 mg/100 ml. Panniculitis secondary to A1AT deficiency can be successfully treated with liver transplantation or intravenous infusion of A1AT.

Female↗
PubMed1997

Regeneration of the olfactory tract following neonatal lesion in rats.

Neuronal regeneration following early postnatal olfactory tract transection (OTS) was investigated in newborn Wistar rats. Olfactory tract lesioned rats were sacrificed at different time periods and the brains processed for Nissl staining. This was used to study the neural cell architecture; fiber tracts (myelinated fibers) were examined with Luxol Fast Blue staining. In addition, a neuronal tracing technique (i.e., retrograde labeling) was employed to study the reestablishment of connections with the target sites following transection of the tract. Degeneration of the olfactory tract was evident at the 7th day following lesion. Regeneration of the tract was not apparent even up to 60 days following transection. However, by 240 days, the olfactory tract had regenerated and the tract fibers had reestablished connection. This was confirmed by retrograde labeling of mitral cells of the olfactory bulb with Fast Blue (FB) injected into the piriform cortex, the target site of these neurons. In this study, we show that mammalian olfactory tract can regenerate spontaneously if the olfactory tract is lesioned neonatally. The results suggest that the olfactory tract is an excellent model to investigate some issues related to central nervous system regeneration.

Amidines↗
PubMed1997

Effect of dexamethasone on ciprofibrate-induced cell proliferation and peroxisome proliferation.

Peroxisome proliferators cause liver cell proliferation in addition to other pleiotropic effects such as peroxisome proliferation and induction of certain peroxisomal and cytosolic enzymes in liver. Since dexamethasone has been shown to inhibit mitogen-induced liver cell hyperplasia, we examined whether dexamethasone inhibits only cell proliferation without affecting peroxisome proliferation induced by peroxisome proliferators such as ciprofibrate. Livers of rats fed a diet containing ciprofibrate (0.025%) with or without added dexamethasone (0.5 mg or 1 mg/kg diet) for 1 week were evaluated for hepatocyte proliferation and peroxisome proliferation. Dexamethasone administration resulted in abrogation of ciprofibrate-induced cell proliferation as shown by bromodeoxyuridine (BrdU) labeling and mitoses counts. The hepatocyte proliferative index measured after administration of a single dose of BrdU was 18.3 +/- 1.1 and 2.3 +/- 0.7% (p < 0.01) in ciprofibrate and ciprofibrate + dexamethasone treated rats, respectively. With multiple injections of BrdU (daily injections for 7 days) the proliferative index was 225 +/- 10 and 183 +/- 2% (p < 0.02), respectively, in these two groups. Interestingly, whereas the levels of peroxisome proliferator-induced Mr 80,000 polypeptide and catalase and peroxisomal bifunctional enzyme, and the corresponding mRNAs and peroxisome volume density were unaffected. These results show that dexamethasone selectively inhibits only cell proliferation without inhibiting the peroxisome proliferation caused by ciprofibrate. This model should be useful for examining the role of cell proliferation versus oxidative stress in peroxisome proliferator-induced hepatocarcinogenesis.

Administration, Oral↗
PubMed1997

Atypical acinar cell foci in human pancreas. Morphological and morphometric analysis.

CONCLUSION: The morphological and quantitative findings of the present study suggest that atypical acinar cell foci are not neoplastic in nature. BACKGROUND: Atypical acinar cell foci (AACF) are rare and unusual lesions in the human pancreas. The biological nature of AACF is poorly understood, and is not clear whether they represent neoplastic or degenerative changes in the acinar cells. METHODS: To further characterize and understand the significance of AACF in relation to acinar cell tumor development, we have examined these lesions by light and electron microscopy and evaluated the growth pattern by measuring cell proliferation and the size of the foci in the pancreas of a 16-yr-old male. RESULTS: The pancreas was grossly unremarkable. AACF were randomly distributed throughout the pancreas, well delineated, and showed minimal variation in sizes. The constituent cells contained uniform nuclei, pale vacuolated cytoplasm, and exhibited low nuclear-cytoplasmic ratio. Electron microscopic examination showed a few zymogen granules and markedly dilated rough endoplasmic reticulum. Proliferative index in AACF (13%) was less than in adjacent uninvolved acinar tissue (19%). Quantitative stereological analysis showed the pancreas to contain approximately 1800 AACF/cm3 with a mean focal diameter of 360 microns.

Adolescent↗
PubMed1997

Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells.

We have identified a neuronal-restricted precursor (NRP) cell that expresses E-NCAM (high polysialic-acid NCAM) and is morphologically distinct from multipotent neuroepithelial (NEP) cells (Kalyani et al., 1997) and spinal glial progenitors (Rao and Mayer-Proschel, 1997). NRP cells self renew over multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin-3 (NT-3) and differentiate in the presence of retinoic acid and the absence of FGF into postmitotic neurons. NRP cells can also be generated from multipotent E10.5 NEP cells. Clonal analysis shows that NRP cells arise from a NEP progenitor that generates other restricted CNS precursors. The NEP-derived NRPs undergo self renewal and can differentiate into multiple neuronal phenotypes. Thus, a direct lineal relationship exists between multipotential NEP cells and more restricted neuronal precursor cells present in vivo at E13.5 in the spinal cord.

Animals↗
PubMed1997

Dehydroepiandrosterone inhibits DNA synthesis of rat hepatocytes induced by partial hepatectomy or mitogen (ciprofibrate).

In a previous study we have shown that dehydroepiandrosterone (DHEA) inhibits hepatocyte DNA synthesis after short-term administration and induces hepatocellular carcinomas after long-term administration in the rat. It is not known whether DHEA is also capable of inhibiting replicative and mitogen-induced DNA synthesis. In the present study, we have evaluated the effect of DHEA on DNA synthesis in the rat liver after partial hepatectomy and mitogen administration. After partial hepatectomy, DHEA significantly inhibited DNA synthesis at 20, 26, 32 and 38 h. Similarly, combined administration of ciprofibrate, a peroxisome proliferator and mitogen, and DHEA also resulted in significant hepatocyte DNA synthesis. However, DHEA did not affect liver enlargement caused by ciprofibrate. This experimental system will serve as useful tool to evaluate the role of cell proliferation in carcinogenesis.

Animals↗
PubMed1996

Flip and flop isoforms of chick brain AMPA receptor subunits: cloning and analysis of expression patterns.

Using oligonucleotide primers, we have amplified and sequenced the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors from the brain of 17-day-old chick embryos. Both flip and flop isoforms of each of these glutamate receptors (GluR) were identified and cloned. Nucleotide comparisons showed that the two isoforms for each chick receptor subtype were 71-78% identical, whereas homologous chick and rat isoforms were 94-98% identical. Reverse transcriptase-polymerase chain reaction and restriction enzyme analysis were employed to identify regional variation in flip and flop levels of each AMPA receptor. Flip isoforms of GluR 1-3 predominated in forebrain, while flop variants of GluR 1-4 were more prevalent in the cerebellum. This differential regional expression suggests that alternative splicing of AMPA receptor subunits contributes importantly to synaptic diversity in chick central nervous systems.

Amino Acid Sequence↗
PubMed1996

Cloning and identification of rat deoxyuridine triphosphatase as an inhibitor of peroxisome proliferator-activated receptor alpha.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that transcriptionally regulate responsive genes by binding to the peroxisome proliferator response elements. Protein(s) interacting with PPAR isoforms (alpha, delta, and gamma) may modulate the PPAR-mediated transcriptional activation. Using a yeast two-hybrid system to screen a rat liver cDNA library, we have identified rat deoxyuridine-triphosphatase (dUTPase, EC 3.6. 1.23) as a PPARalpha-interacting protein. This cDNA encodes a polypeptide of 203 amino acids; the C-terminal 141-amino acid segment of this protein corresponds to the full-length human enzyme, which exhibits 92% identity with human dUTPase; the N-terminal extra 62-amino acid residue region is arginine-rich. In vitro binding assays indicate that rat dUTPase interacts with all three isoforms of mouse PPAR, but not with retinoid X receptor and thyroid hormone receptor. Interaction of PPARalpha with dUTPase is with the N-terminal 62-amino acid segment of rat dUTPase. Full-length rat dUTPase prevents PPAR-retinoid X receptor heterodimerization resulting in an inhibition of PPAR activity in a ligand-independent manner. Immunostaining of human kidney tsA201 cells, transiently expressing dUTPase showed that this protein is present predominantly in the cytoplasm but translocates into the nucleus with PPARalpha when PPARalpha is coexpressed with dUTPase. Northern blot hybridization shows that rat dUTPase is encoded by an abundant 1kilobase mRNA species present in all rat tissues. The identification of dUTPase as a PPAR-interacting protein suggests a possible link between tumorigenic peroxisome proliferators and the enzyme system involved in the maintenance of DNA fidelity.

Amino Acid Sequence↗
PubMed1996

Transformation of epithelial cells stably transfected with H2O2-generating peroxisomal urate oxidase.

Peroxisome proliferators, a group of structurally diverse nongenotoxic agents, induce predictable pleiotropic responses in liver, including the development of liver tumors in rats and mice. These agents transcriptionally activate the three genes of the peroxisomal beta oxidation enzyme system by interacting with the peroxisome proliferator-activated receptor(s). It has been proposed that H2O2 generated by the peroxisomal beta oxidation system leads to DNA damage and neoplastic transformation. Consistent with this hypothesis is that cells stably transfected with H2O2-generating peroxisomal fatty acyl-CoA oxidase cDNA, which encodes the first and rate-limiting enzyme of the beta oxidation system, undergo transformation in the presence of a fatty acid substrate. To test whether H2O2 generated by other peroxisomal oxidases can also lead to transformation, a full-length cDNA encoding rat urate oxidase (UOX), which oxidizes uric acid to allantoin and in the process generates H2O2, was introduced into African green monkey kidney cells (CV-1 cells) under the control of constitutively active human peroxisomal fatty acyl-CoA oxidase gene promoter. Five stably transfected CV-1 cell lines expressing recombinant rat UOX were isolated in which the recombinant protein was targeted to peroxisomes and formed crystalloid structures or cores similar to those present in rat liver peroxisomes. Increased levels of H2O2 were found when cells stably expressing UOX were exposed to the substrate uric acid. These five clones, designated A-U1 to A-U5, exhibited anchorage-independent growth, as demonstrated by the formation of transformed colonies in soft agar in proportion to the duration of exposure to uric acid. These transformants exhibited clonal growth under serum-deprived conditions. One of these transformed cell lines, the A-U3 cell line, was evaluated for tumorigenicity by s.c. injection in nude mice. All five mice injected with transformed A-U3 cells developed adenocarcinomas, but no tumors developed in mice injected with control CV-1 cells or cells stably expressing UOX that were not exposed to uric acid. These results provide further evidence indicating that sustained overexpression of a peroxisomal H2O2-generating oxidase causes cell transformation.

Animals↗