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M R Lloyd

Publications and source records attributed to M R Lloyd.

12 recordsLinked to original sources
PubMed1989

Calmodulin quantification and immunolocalization in developing embryonic orofacial tissue.

Temporally and quantitatively coordinated synthesis of cyclic adenosine monophosphate appears to be critical for normal development of the mammalian secondary palate. Calmodulin has been implicated as being involved in mediating the activity of a number of fundamental calcium-regulated intracellular enzyme systems including phosphodiesterases, adenylate cyclase, and a variety of kinases, all of which may regulate or be regulated by intracellular cAMP. Calmodulin levels were thus quantified, and endogenous calmodulin was immunolocalized in developing palatal tissue in vivo and in embryonic palatal mesenchymal cells in vitro. Endogenous palatal calmodulin levels, determined by radioimmunoassay, showed little variation during the period of murine palatal ontogenesis and averaged 0.23 ng/micrograms protein. Murine palate mesenchymal cells in monolayer, either in logarithmic growth or at confluency, contained similar levels of calmodulin. In palate mesenchymal cells in primary culture, specific anti-calmodulin staining was confined to the cell cytoplasm and was concentrated in the perinuclear region. Since immunostaining for calmodulin appeared to be associated with discrete cytoplasmic filaments, distribution of actin and tubulin were investigated. Immunostaining for tubulin in these cells was also localized to the perinuclear region, while immunolocalization of actin showed staining patterns, reflective of stress fibers, which were quite different from those seen for calmodulin. Immunostaining was seen in vivo in all regions of the palatal epithelium with superficial peridermal cells staining most intensely. Specific immunostaining was also evident in palatal mesenchyme, where a pericellular distribution was seen. Staining patterns were similar throughout the period of palatal ontogeny. In addition, a sharply defined localization of calmodulin to cartilagenous extracellular matrix was noted. This study provides a useful initial approach toward understanding the role calmodulin may play in embryonic orofacial development.

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↗
PubMed1985

Effect of epidermal growth factor on synthesis of prostaglandins and cyclic AMP by embryonic palate mesenchymal cells.

The influence of epidermal growth factor (EGF) on the ability of murine embryonic palate mesenchymal (MEPM) cells to be stimulated to synthesize cAMP and prostaglandins was investigated. Preincubation of MEPM cells with EGF enhanced, in a dose-dependent fashion, (1) the responsiveness of MEPM cells to prostaglandin E1-induced elevation of intracellular levels of cAMP, and (2) the responsiveness of cells to calcium ionophore (A23187) and melittin-induced synthesis of prostaglandins E2 and F2 alpha. Hormonal responsiveness of MEPM cells to EGF, prostaglandins and cAMP has been implicated as being involved in controlling various aspects of normal oro-facial development. We show here that EGF can potentiate hormonal responsiveness of these cells and thus allows consideration of EGF as a factor which may modulate hormonally regulated craniofacial growth and differentiation.

Animals↗
PubMed1985

Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities.

During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates.

Alprostadil↗
PubMed1979

Follow-up evaluation of treatment of drug abuse during 1969 to 1972.

A sample of 3,131 persons from approximately 25,000 admitted to drug abuse treatment programs in the Drug Abuse Reporting Program during 1969-1972 were followed up in 1975-1976. Treatment groups included methadone hydrochloride maintenance, therapeutic community, outpatient drug free, outpatient detoxification, and a comparison group that completed intake but did not enter treatment. Outcome criterion measures (drug use, employment, criminality, and treatment readmissions) based on the first year after treatment were more favorable in the methadone maintenance, therapeutic community, and outpatient drug-free groups than in the outpatient detoxification and intake-only groups. Evaluation of differential outcomes and their relationships with pretreatment and during-treatment measures within each group indicated that pretreatment criminal history, during-treatment performance, and length of time in treatment were significantly related to posttreatment outcomes.

Adult↗
PubMed1979

Client evaluations of drug abuse treatment in relation to follow-up outcomes.

A national sample of drug treatment clients admitted to the Drug Abuse Treatment Program (DARP) during 1969--1972 was followed approximately 5 years after admission. Subjective evaluations of treatment were compared for clients in methadone maintenance, therapeutic community, outpatient drug-free, and outpatient detoxification treatment programs, as well as a comparison group of clients who completed intake but received no treatment. These evaluations were also analyzed in relation to client background and treatment performance measures and post-treatment outcomes. The overall evaluation of DARP treatments by former clients was generally favorable and was highest for the DARP therapeutic community. Over two-thirds of the combined treatment sample indicated satisfaction with the treatment they received and a willingness to recommend it to others. Favorable evaluation was also positively related to during-treatment performance and tenure in DARP treatment as well as to behavioral outcomes after leaving treatment.

Adolescent↗
PubMed1978

Alcohol and illicit drug use: follow-up study of treatment admissions to DARP during 1969-1971.

The present study was based on follow-up data on 1409 persons interviewed 4 to 6 years after admission to drug treatment in the Drug Abuse Reporting Program (DARP). The admissions to DARP occurred in 1969-1971, and for most persons the follow-up data included 3 or more years after termination of DARP treatment. The study focused on variations in alcohol consumption associated with post-DARP drug use and treatment status. The results indicated that use of alcohol and non-opioid drugs (particularly marijuana) tended to be correlated, but that this was not true of opioid drugs. For a small segment of the sample, evidence suggested that substitution of use may have occurred between alcohol and opioid drugs. Also, persons with post-DARP drug treatment tended to use less alcohol than persons without treatment. Interpretations and implications of these findings are discussed.

Adult↗
PubMed1976

A national follow-up study to evaluate the effectiveness of drug abuse treatment: a report on cohort 1 of the DARP five years later.

Preliminary findings for the first cohort of a national follow-up study of drug users admitted to treatment in the Drug Abuse Reporting Program (DARP) during 1969-1971 are reported. From a sample of 1,853 selected for follow-up, 87% were located and interviews were completed for 77%. Gross tabulations are shown comparing several outcome measures based on pre-DARP treatment, the posttreatment period, and for the 2 months before interview. Sixty-one percent of all patients returned to treatment at some time after DARP termination. Decreases in drug use, expecially of opioids, were substantial and were generally accompanied by improvements in other outcome measures as well. Plans for more detailed and informative analysis of these and related DARP data are addressed.

Ethnicity↗
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↗