Biomedical subjects

M M Pisano

Publications and source records attributed to M M Pisano.

30 records · Page 2Linked to original sources

PubMed1995

Patterns of cyclic AMP-dependent protein kinase gene expression during ontogeny of the murine palate.

Normal growth and differentiation of embryonic palatal tissue depends on regulated levels of intracellular cAMP. Cyclic AMP-dependent protein kinases (PKA) act to mediate the biological activities of cAMP. PKA isozyme protein profiles demonstrate a clear pattern of temporal alterations in embryonic palatal tissue during its development. In order to ascertain the molecular basis for changing PKA isozyme profiles during palatal ontogeny, the spatial and temporal expression of mRNAs for regulatory (RI alpha, RII alpha, and RII beta) and catalytic (C alpha) subunits of PKA was examined. RNA extracted from murine embryonic palatal tissue (days 12-14 of gestation) was examined by Northern blot analysis. Significant levels of constitutively expressed RI alpha and C alpha mRNA were seen on all days of gestation examined. RI alpha transcripts were substantially less abundant in palate mesenchymal cells in vitro than in palatal tissue in vivo. Levels of RII alpha and RII beta mRNA were highest on gestational day (GD) 12, a period characterized by pronounced palatal tissue growth. In addition, patterns of tissue distribution of RII beta, not previously described, were examined in the developing embryonic palate. A dramatic developmental shift in tissue distribution of RII beta was seen. The isozyme was evenly distributed between palatal epithelial and mesenchymal cells on GD 12 but by GD 14, RII beta was predominantly localized to palatal epithelial cells. Direct activation of adenylate cyclase with forskolin in murine embryonic palate mesenchymal (MEPM) cells resulted in an increase in RII alpha mRNA levels but had no effect on steady state levels of RII beta or C alpha mRNA. In addition, elevation of intracellular levels of cAMP resulted in a shift in the transcriptional profile of RI alpha mRNAs. Results of this study document specific patterns of expression for the genes encoding the various cAMP-dependent protein kinase regulatory and C alpha subunits in murine embryonic palatal tissue. In addition, we have demonstrated adaptational changes of this kinase in MEPM cells in response to conditions of increased intracellular levels of cAMP.

Animals↗

PubMed1995

Molecular cloning, expression, and developmental characterization of the murine retinoblastoma-related gene Rb2/p130.

The product of the retinoblastoma-related human gene Rb2/p130 is highly homologous with the product of the retinoblastoma tumor suppressor gene (pRb) and Rb-related p107. this homology is shared mainly in the pocket domain, a region that seems to play a key role in the functions of these proteins. Here we report the molecular cloning and initial characterization of the cDNA encoding the murine homologue of the human Rb2/p130 gene product. The 4.8-kb cDNA encodes a protein of 1125 amino acids that shows 90% indentity to that of the human protein. The Rb2/p130 mRNA is found to be expressed in all of the adult mouse tissues examined, with the highest level being detected in kidney and skeletal muscle. For the protein characterization, we used a polyclonal antibody raised against the COOH terminus of the human Rb2/p130 protein that also recognizes the mouse protein. In developing mouse embryos, the Rb2/p130 protein is expressed as early as day 10 of gestation and reached a peak of expression around day 13 of gestation, implying a developmental regulation of the Rb2/p130 gene in murine ontogeny.

3T3 Cells↗

PubMed1994

Regulation of TGF beta 3 gene expression in embryonic palatal tissue.

The TGF beta family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGF beta exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGF beta s. We therefore examined the expression of mRNAs encoding TGF beta 1, TGF beta 2, and TGF beta 3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGF beta 1 (2.5 kb), two transcripts for TGF beta 2 (4.4 kb, 6.0 kb), and one transcript for TGF beta 3 (3.5 kb). Although steady-state levels of TGF beta 1 mRNA showed no changes during development of the palate, TGF beta 2 mRNA levels were maximal on both GD13 and GD14 and TGF beta 3 mRNA levels transiently increased on GD 13. In addition, levels of TGF beta 3 mRNA seemed much higher than either TGF beta 1 or TGF beta 2. both TGF beta 1 and TGF beta 2 were able to increase, in a dose-related manner, the expression of TGF beta 3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGF beta 3 mRNA even in the presence of TGF beta 1 or TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Ethanol effects on embryonic craniofacial growth and development: implications for study of the fetal alcohol syndrome.

Fetal alcohol syndrome (FAS), which is brought about by maternal consumption of ethanol during pregnancy, is a major public health problem. To gain understanding of the etiology of this condition, a number of teratological studies have been performed in different animal systems to develop an animal model for FAS. The C57BL/6J mouse strain has been described as susceptible to the teratogenic effects of ethanol, whereas the ICR (CD-1) strain is considered relatively insensitive. We have compared the effects of ethanol on DNA and protein synthesis in cultured embryonic palate mesenchymal cells from both strains to determine if the reported differential sensitivity to ethanol is reflected in differences in ethanol's effects on cell behavior. Chronic exposure to 200 mM ethanol for 48 hr had a strong inhibitory effect on DNA synthesis in palate cells derived from both the C57BL/6J and ICR strains and a significant effect on protein synthesis in C57BL/6J palate cells. When we attempted to verify strain differences in susceptibility to ethanol teratogenesis, we were not able to observe an increased incidence of birth defects due to ethanol in either strain. High doses of ethanol (5.8 g/kg, administered by intraperitoneal injection on gestational day 8) resulted in death in both C57BL/6J and ICR mice. A lower dose (4.8 g/kg) caused decreased fetal weight and increased resorption in both strains, but did not bring about FAS-like craniofacial dysmorphology in either strain. It appears, therefore, that whereas ethanol can significantly affect the behavior of cells derived from craniofacial tissue, these effects cannot be correlated with sensitivity to ethanol teratogenesis in the mouse system.

Animals↗

PubMed1991

Genomic cloning, complete nucleotide sequence, and structure of the human gene encoding the major intrinsic protein (MIP) of the lens.

Major intrinsic protein (MIP, also called MP26) is the predominant fiber cell membrane protein of the ocular lens. MIP has been suggested to play a role in cell-cell communication in the lens. Its expression is tissue-specific and developmentally regulated. We have isolated and characterized the human gene encoding MIP and report here its genomic structure and entire nucleotide sequence. The gene is 3.6 kb, contains four exons separated by introns ranging in size from 0.4 to 1.6 kb, and is present in single copy per haploid human genome. Primer extension of human lens RNA indicates that transcription of the gene initiates from a single site 26 nt downstream from the TATA box. Three complete Alu repetitive elements are found in tandem in the 5'-flanking region of the gene, and a single complete Alu sequence is present in the third intron. The interspecies comparisons of the MIP gene coding sequence and homologies to other members of a putative transmembrane channel protein superfamily are also discussed.

Amino Acid Sequence↗

PubMed1991

Tandem sequence repeats in transmembrane channel proteins.

The flow of ions and small molecules out of and between cells is mediated by various classes of transmembrane proteins. One group of putative channel proteins, including the abundant lens protein MIP, is widely distributed from prokaryotes to vertebrates. This article suggests that these proteins contain a structural twofold repeat and may have arisen by gene duplication. Such a model has implications for the tertiary structures of these important proteins.

Amino Acid Sequence↗

PubMed1991

Lens protein gene expression: alpha-crystallins and MIP.

The crystallin genes encode the major soluble proteins of the lens. Some of the crystallin genes are expressed exclusively in the lens while others are also expressed in different tissues. The two alpha-crystallin genes, alpha A and alpha B, differ in their tissue specificity. Transcription of the alpha A-crystallin gene occurs only in the lens, while the alpha B-crystallin gene is also expressed in other tissues, including heart, skeletal muscle, kidney, lung and brain. MIP (also called MP26), the major intrinsic protein of the lens fiber membranes, is also expressed exclusively in the lens. Correct expression of both alpha-crystallin and MIP are required for normal lens function. Here we review our studies on the molecular basis of expression of the alpha-crystallin and MIP genes in the lens. The 5' flanking sequences containing the initiation site of transcription of the alpha A-crystallin, alpha B-crystallin and MIP genes were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the expression of this reporter gene was studied in transient assays and transgenic mice. DNA sequences flanking the 5' end of the alpha A-crystallin gene contain regulatory elements responsible for the lens-specific expression and developmental regulation of the CAT gene in transgenic mice. Interestingly, although some of the murine alpha A-crystallin regulatory sequences are conserved in the human and chicken genes, different functional regulatory elements appear to control the expression of the murine and chicken alpha A-crystallin genes. The 5' flanking sequence of the alpha B-crystallin gene preferentially directs expression of the CAT gene to the lens and to skeletal muscle. Different regulatory elements of the alpha B-crystallin gene appear to be responsible for its transcription in various tissues. The 5' flanking sequence of the MIP gene also contains regulatory elements that direct expression of the CAT gene to lens cells; these sequences are not functional in transfected non-lens cells and are different from the cis regulatory elements controlling alpha-crystallin gene expression. The multiplicity of cis-regulatory elements controlling the transcription of these three genes indicates the complexity of the mechanisms that regulate gene expression in the lens.

Animals↗

PubMed1989

Characterization of soluble cyclic adenosine monophosphate-dependent protein kinase isozymes in murine embryonic palatal tissue.

Certain hormonal primary messengers identified in the mammalian palate during its ontogeny transmit information to the interior of the cell via transmembrane signaling systems that control the production of the secondary messenger cyclic adenosine monophosphate. The singular role of intracellular cyclic AMP is to activate cAMP-dependent protein kinases (cAMP-dPK). cAMP-dPK were thus identified and characterized in the developing murine embryonic palate. Incubation of cytosolic fractions of embryonic palatal tissue with cAMP resulted in a dose-dependent increase in the cAMP-dPK activity ratio. A transient elevation of basal cAMP-dPK was seen during the period of palatal ontogeny that corresponded temporally with a previously demonstrated transient elevation of palatal basal cAMP levels. Fractions of embryonic palatal tissue cytosols derived by diethylaminoethyl (DEAE)-Sephacel chromatography were analyzed for phosphotransferase activity and for [3H]-cAMP binding to the regulatory (R) subunits of cAMP-dPK. Such analyses revealed two peaks of activity on day 13 of gestation. Based on the salt concentration at which the material in these peaks eluted from DEAE, its ability to cochromatograph with authentic cAMP-dPK isozymes, its molecular weight as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis, and the ability of the material to be photoaffinity labeled with [3H]-8-azidoadenosine 3',5' cyclic phosphate, types I and II cAMP-dPK were identified. Regulatory subunits of cAMP-dPK were characterized by the binding of [3H]-cAMP to cytosolic fractions of embryonic palatal tissue. Such binding was saturable (Bmax = 1,096 fmol/mg protein) and of high affinity (Kd = 7 nM). Only cAMP and cyclic guanosine monophosphate competed in a dose-related manner with [3H]-cAMP for binding to R subunits of cAMP-dPK. Adenosine, cTMP, and adenosine triphosphate, at doses up to 10(-4) M, did not compete for binding. Temporal analysis of binding data indicated that the number of binding sites transiently decreased during day 13 of gestation. Characterization of cAMP-dPK in tissue derived from the developing mammalian palate allows consideration of cAMP-dPK as a key regulatory enzyme capable of transducing hormonally elevated intracellular levels of cAMP into metabolic responses during orofacial ontogenesis.

Animals↗

PubMed1987

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis.

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.

Animals↗

PubMed1986

Catecholamine modulation of embryonic palate mesenchymal cell DNA synthesis.

Development of the mammalian embryonic palate depends on the precise temporal and spatial regulation of growth. The factors and mechanisms underlying differential growth patterns in the palate remain elusive. Utilizing quiescent populations of murine embryonic palate mesenchymal (MEPM) cells in vitro, we have begun to investigate hormonal regulation of palatal cell proliferation. MEPM cells in culture were rendered quiescent by 48 hr serum deprivation and were subsequently released from growth arrest by readdition of medium containing 10% (v/v) serum. The progression of cells into S-phase of the cell cycle was monitored by autoradiographic analysis of tritiated thymidine incorporation. Palate mesenchymal cell entry into S-phase was preceded by a 6- to 8-hr prereplicative lag period, after which time DNA synthesis increased and cells reached a maximum labeling index by 22 hr. Addition of 10 microM isoproterenol to cell cultures at the time of release from growth arrest lengthened the prereplicative lag period and delayed cellular entry into S-phase by an additional 2 to 4 hr. The rate of cellular progression through S-phase remained unaltered. The inhibitory effect of isoproterenol on the initiation of MEPM cell DNA synthesis was abolished by pretreatment of cells with propranolol at a concentration (100 microM) that prevented isoproterenol-induced elevations of cAMP. Addition of PGE2 to cell cultures, at a concentration that markedly stimulates cAMP formation, mimicked the inhibitory effect of isoproterenol on cellular progression into S-phase. These findings demonstrate the ability of the beta-adrenergic catecholamine isoproterenol to modulate MEPM cell proliferation in vitro via a receptor-mediated mechanism and raise the possibility that the delayed initiation of DNA synthesis in these cells is a cAMP-dependent phenomenon.

Animals↗

PubMedDate not supplied

Cyclic AMP-dependent protein kinase in human embryonic palate mesenchymal cells.

Growth and differentiation of cells derived from the embryonic palate are critically dependent on the intracellular cAMP-mediated signal transduction pathway. Human embryonic palate mesenchymal (HEPM) cells have been widely used to examine the effect of teratogens on palatal tissue growth and differentiation, as well as a prescreen for environmental teratogens. This study examined responsiveness of HEPM cells to agents known to stimulate adenylate cyclase, characterized cAMP-dependent protein kinases (cAMP-dPK) (EC 2.7.1.37) and investigated to what extent HEPM cells reveal adaptational responses to cAMP at the level of cAMP-dependent protein kinase. HEPM cells exhibited a total cell cycle transit time of approximately 22 h and responded maximally, when confluent, to prostacyclin (PGI2), prostaglandin E2 (PGE2), and isoproterenol with time- and dose-dependent increases in intracellular levels of cAMP. The order of sensitivity to hormonal activation of adenylate cyclase was PGE2 > isoproterenol > PGI2. Basal cAMP-dependent protein kinases activity was 0.184 fmol phosphate transferred from ATP to histone per microgram protein per minute under conditions where endogenous phosphatases did not significantly affect protein phosphorylation. Regulatory subunits of cAMP-dPK in HEPM cells were characterized by the binding of [3H]cAMP to cytosolic fractions. Specific binding was saturable at approximately 50 nM indicating the presence of binding sites that are finite in number. Calculation of half-maximal binding yielded an estimated Kd of 25 nM indicating the presence of high affinity binding sites. Cyclic AMP-dPK regulatory subunits were also photoaffinity labeled with 8-N3-[32P]-cAMP, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radiolabeled bands visualized by autoradiography. Photoactivated incorporation of 8-N3-[32P]cAMP was detected into two proteins of molecular weight (M(r)) 45,000 and M(r) 51,000 representing, respectively, the RI alpha and RII beta subunits of cAMP-dPK. Binding of [32P]8-azido cAMP to proteins of M(r) 45,000 (RI alpha) and M(r) 51,000 (RII beta) was increased in response to elevation of intracellular cAMP via inhibition of its breakdown with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by direct activation of adenylate cyclase with forskolin. HEPM cells thus revealed adaptational responses to cAMP at the level of cAMP-dependent protein kinase. Characterization of the cAMP signal transduction pathway in HEPM cells, derived from embryonic palatal tissue which is critically dependent on this pathway for normal development, may provide information fundamental to a clear understanding of cellular events involved in palatal ontogeny. These results highlight several important differences between HEPM cells and murine embryonic palate mesenchymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMedDate not supplied

Transmembrane and intracellular signal transduction during palatal ontogeny.

Investigations of the pathogenesis of cleft palate have been hampered by the complexity of normal craniofacial ontogenesis and by an incomplete understanding of mechanisms involved in normal palate development. This article deals with cellular aspects of the developing craniofacial region and discusses the facts and problems in the field as they relate to the normal development of the secondary palate, with emphasis on the regulation of hormone- and growth factor-induced transmembrane signalling and intracellular signal transduction.

Animals↗