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H W Davis

Publications and source records attributed to H W Davis.

At least 37 records · Page 2Linked to original sources
PubMed1992

Insulin-stimulated phosphorylation of calmodulin.

Calmodulin is phosphorylated in vitro by the insulin-receptor tyrosine kinase and a variety of serine/threonine kinases. Here we report that insulin stimulates the phosphorylation of calmodulin on average 3-fold in intact rat hepatocytes. Although calmodulin is constitutively phosphorylated, insulin increases phosphate incorporation into serine, threonine and tyrosine residues. We demonstrate that casein kinase II, an insulin-sensitive kinase, phosphorylates calmodulin in vitro on serine/thyronine residues (Thr-79, Ser-81, Ser-101 and Thr-117). The ability of the insulin receptor to phosphorylate calmodulin that has been pre-phosphorylated by casein kinase II is enhanced up to 35-fold, and the sites of phosphorylation on calmodulin are shifted from tyrosine to threonine and serine. These observations, obtained with a new specific monoclonal antibody to calmodulin, confirm that insulin stimulates calmodulin phosphorylation in intact cells. The observation that calmodulin is phosphorylated in vivo, coupled with the recent demonstration that phosphocalmodulin exhibits altered biological activity, strongly suggests that phosphorylation of calmodulin is a critical component of intracellular signalling.

Amino Acid Sequence↗
PubMed1992

Phosphorylation by casein kinase II alters the biological activity of calmodulin.

Calmodulin is the major intracellular Ca(2+)-binding protein, providing Ca(2+)-dependent regulation of numerous intracellular enzymes. The phosphorylation of calmodulin may provide an additional mechanism for modulating its function as a signal transducer. Phosphocalmodulin has been identified in tissues and cells, and calmodulin is phosphorylated both in vitro and in intact cells by various enzymes. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreases its ability to activate both myosin-light-chain kinase and cyclic nucleotide phosphodiesterase. For myosin-light-chain kinase the primary effect is an inhibition of the Vmax. of the reaction, with no apparent change in the concentration at which half-maximal velocity is attained (K0.5) for either Ca2+ or calmodulin. In contrast, for phosphodiesterase, phosphorylation of calmodulin significantly increases the K0.5 for calmodulin without noticeably altering the Vmax. or the K0.5 for Ca2+. The higher the stoichiometry of phosphorylation of calmodulin, the greater the inhibition of calmodulin-stimulated activity for both enzymes. Therefore the phosphorylation of calmodulin by casein kinase II appears to provide a Ca(2+)-independent mechanism whereby calmodulin regulates at least two important target enzymes, myosin-light-chain kinase and cyclic nucleotide phosphodiesterase.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation.

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.

Animals↗
PubMed1992

Identification, partial purification, and characterization of two guanosine triphosphate-binding proteins associated with insulin receptors.

We have previously suggested that at least two different G-proteins are involved in mediating insulin receptor functions. Here we identify and partially purify two G-proteins with apparent molecular masses of 41 and 67 kilodaltons (kDa) that interact with insulin receptors in rat adipocytes and human placenta. Treatment of isolated rat adipocytes with insulin inhibited pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa G-protein in subsequently isolated plasma membranes by 30.2 +/- 3.0% and in partially purified insulin receptor preparations by 35.6 +/- 5.7%. There was no associated decrease in the concentration of the 41-kDa G-protein in the plasma membranes, as determined by immunoblot with a common G alpha antibody. The common G alpha antibody also recognized a 67-kDa protein in the plasma membranes, the concentration of which was not affected by insulin. However, the 67-kDa protein was enriched in partially purified solubilized insulin receptor preparations. Two similar, 41- and 67-kDa G-proteins were identified in the wheat germ-purified insulin receptor preparations obtained from human placenta. Removal of these two G-proteins from insulin receptor preparations results in loss of the ability of insulin to stimulate receptor kinase activity. Addition of a fraction enriched with 41- and 67-kDa G-proteins to the G-protein-depleted insulin receptor restores the insulin sensitivity of the insulin receptor kinase activity. Furthermore, addition of G-protein-depleted insulin receptors to the fraction containing partially purified 41- and 67-kDa G-proteins enhances pertussis toxin-catalyzed ADP-ribosylation of the 41-kDa G-protein. These results indicate that either the 41- or 67-kDa G-protein, or both, interact with the insulin receptor mediating insulin receptor kinase activity. Such mutual interaction and regulation between the insulin receptor and G-proteins could be an important component of the signal transduction mechanism for insulin.

Adenosine Diphosphate Ribose↗
PubMed1991

Development and DNA polymerase activities in cultured preimplantation mouse embryos: comparison with embryos developed in vivo.

Embryos from superovulated female mice that developed in vitro from the two-cell stage were compared with in vivo embryos with respect to yield of blastocytes, number and types of cells, morphology in histologic section, and DNA polymerase activities. Significantly more embryos developed into blastocytes in vitro (93%) than in vivo (18%). Inner cell mass (ICM) cells comprised approximately 30% of total cells in late morula/early blastocyst stage embryos developed either in vitro or in vivo. However, the in vitro embryos developed approximately half the number of total cells as in vivo embryos, did not develop endoderm, and did not develop abembryonic trophoblast cells with morphologic characteristics of late preimplantation in vivo embryos. DNA-dependent DNA polymerase activities in in vitro embryos decreased in correspondence with the decrease in cell number resulting in per cell levels comparable to in vivo embryos. In contrast, the poly (A).oligo(dT)-dependent DNA polymerase activity was the same in embryos developing either in vitro or in vivo, indicating different regulatory mechanisms for the two enzyme activities. A variety of nutrients and growth factors in the culture medium did not increase cell numbers or DNA polymerase activities in embryos cultured for 3 days; extending the culture an additional 24 hours resulted in a loss of ICM cells and decreases in both DNA polymerase activities. These results show that the retarded growth of embryos in vitro is equally distributed between ICM and trophoblast, is not reversed by culture conditions that include serum growth factors, and is not due to decreased cellular levels of DNA polymerase activities.

Animals↗
PubMed1991

Pediatric emergency department complaints: a three-year analysis of sources and trends.

STUDY OBJECTIVE: To identify the incidence and major causes of patient and parental complaints in a pediatric emergency department. DESIGN: Retrospective analysis of complaints received regarding patients seen between January 1987 and December 1989. SETTING: ED of Children's Hospital of Pittsburgh. PARTICIPANTS: All complaints received during the three-year period. INTERVENTIONS: Complaints were reviewed for reason, validity, and location at which patient was seen. MEASUREMENTS AND RESULTS: One hundred seventy-six complaints from a total of 154,648 ED visits yielded a frequency of 1.1 complaints per 1,000 patient visits. Main reasons for dissatisfaction were misdiagnosis, billing, and inadequate treatment; 49% of complaints were judged valid. There were 0.69 complaints per 1,000 patient visits in the nonurgent medical portion of the ED. Patients seen emergently (critical care and trauma) had a significantly lower complaint frequency of 0.08 per 1,000 patient visits (P less than .001 by chi 2 analysis). CONCLUSION: Assessment of ED complaints is useful to highlight areas of patient dissatisfaction and develop plans for improving patient care.

Accounts Payable and Receivable↗
PubMed1991

Dental needs assessment survey: three Florida counties.

Dental needs assessment surveys were completed in three Florida counties (1987 - Lee County), (1988 - Pasco County) and (1989 - St. Lucie County). The data collected were then compared with the results from the national caries prevalence surveys in 1979-80 and 1986-87, by individual county and with the combined three-county total. Visual tactile examinations were done using the Radlike criteria for the DMFS index and the WHO Treatment Needs Index. Schools were selected based on the percent of students enrolled in the free or reduced lunch program and by geographic distribution. Although there were age group differences within individual counties, the combined data showed every age group except the seven year olds at a lower DMFS mean level than the data from the 1986-87 national survey for Region IV. The differences range from -.20 surfaces in 8-year-olds to -.57 surfaces in 13-year-olds. None of the differences were statistically significant from the national surveys. This indicates that these small scale surveys are a reliable method for needs assessment.

Adolescent↗
PubMed1990

Insulin receptor function is inhibited by guanosine 5'-[gamma-thio]triphosphate (GTP[S]).

The regulatory role of GTP-binding proteins (G-proteins) in insulin receptor function was investigated using isolated insulin receptors and plasma membranes from rat adipocytes. Treatment of isolated insulin receptors with 1 mM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) inhibited insulin-stimulated phosphorylation of the beta-subunit, histone Hf2b and poly(GluNa4,Tyr1) by 22%, 65% and 65% respectively. Phosphorylation of calmodulin by the insulin receptor kinase was also inhibited by 1 mM-GTP[S] both in the absence (by 88%) and in the presence (by 81%) of insulin. In the absence of insulin, 1 mM-GTP had the same effect on calmodulin phosphorylation as 1 mM-GTP[S]. However, when insulin was present, GTP was less effective than GTP[S] (41% versus 81% inhibition). Concentrations of GTP[S] greater than 250 microM are necessary to inhibit phosphorylation. Although these concentrations are relatively high, the effect of GTP[S] is not due to competition with [32P]ATP for the insulin receptor kinase since (1) other nucleotide triphosphates did not inhibit phosphorylation as much as did GTP[S] (or GTP) and (2) the Vmax of the ATP-dependent kinase reaction was decreased in the presence of GTP[S]. GTP[S] (1 mM) also inhibited insulin binding to isolated receptors and plasma membranes, by 80% and 50% respectively. Finally, an antibody raised to a peptide sequence common to the alpha-subunits of G-proteins Gs, Gi, Go and transducin detected G-proteins in plasma membranes but failed to detect them in the insulin receptor preparation. These results indicate that GTP inhibits insulin receptor function, but does so through a mechanism that does not require a conventional GTP-binding protein.

Adipose Tissue↗
PubMed1990

Modulation of guanine nucleotide effects on the insulin receptor by MgCl2.

Insulin binding to partially purified rat adipocyte insulin receptors is inhibited approximately 40-60 percent by 1 mM GTP-gamma-S in the presence of 2 mM MgCl2. However, in the presence of 10 mM MgCl2, GTP-gamma-S does not inhibit binding. Increasing MgCl2 from 0.5 to 10 mM enhances the phosphorylation of calmodulin catalyzed by the insulin receptor but also reduces the inhibition seen with 500 microM GTP-gamma-S. The reversal of the GTP-gamma-S-induced inhibition of calmodulin phosphorylation by high concentrations of MgCl2 appears to be due to an effect on the calmodulin molecule since MgCl2 has little effect on the inhibition of phosphorylation of histone Hf2b or poly (Glu4, Tyr1) induced by GTP-gamma-S. Our data suggest that there are at least two GTP-binding proteins associated with the insulin receptor, one that regulates insulin binding and is modulated by MgCl2 and one that regulates substrate phosphorylation and/or receptor-substrate coupling and is not altered by MgCl2.

Adipose Tissue↗
PubMed1989

Increases in plasma concentrations of a prostaglandin metabolite in acute airway obstruction.

Plasma concentrations of a stable prostaglandin F2 alpha metabolite were measured by radioimmunoassay during and after recovery from acute airway obstruction in 15 infants. Mean (SEM) metabolite concentrations (ng/l) in plasma obtained both before (1033 (418)) and after (1470 (413)) initial treatment for airway obstruction were significantly higher than those obtained from the same subjects after resolution of the obstruction--25.5 (6.6)--and those obtained from two comparison groups. Infants positive for respiratory syncytial virus (mean 1122 (227)) had significantly higher concentrations than those who were negative (207.6 (46)). Additionally, seven subjects with a history of recurrent wheezing after resolution of airway obstruction had a significantly higher mean level (3500 (1400)) during attacks of airway obstruction than those without (600 (100)). These data suggest that prostaglandin F2 alpha mediates respiratory inflammation in airway obstruction and that trials of specific anti-inflammatory agents for the treatment of airway obstruction may be warranted.

Airway Obstruction↗
PubMed1988

Prostaglandin E2 and thromboxane B2 release from human monocytes treated with bacterial lipopolysaccharide.

We investigated the capacity of counterflow-isolated human monocytes to independently synthesize thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) when stimulated with bacterial lipopolysaccharide (LPS). Independent metabolism was confirmed by establishing different specific activities (dpm/ng) of TxB2 and PGE2 released from LPS-treated cells. For metabolites released during the initial 2-hr treatment period, the specific activity of PGE2 was approximately threefold higher than that of TxB2 regardless of labeling with [3H]arachidonic acid (AA) or [14C]AA. Cells that were pulse-labeled for 2 hr with [3H]AA demonstrated a decreasing PGE2 specific activity over 24 hr, whereas the TxB2 specific activity remained unchanged. In contrast, cells continuously exposed to [14C]AA demonstrated an increasing TxB2 specific activity that approached the level of PGE2 by 24 hr. These results suggest the presence of at least 2 cyclooxygenase metabolic compartments in counterflow-isolated monocytes. Although freshly isolated monocytes have been reported to contain variable numbers of adherent platelets, additional experiments demonstrated that counterflow-isolated platelets are not capable of releasing elevated levels of TxB2 or PGE2 when treated with LPS. It is proposed from these findings that at least two subsets of monocytes exist in peripheral blood that can be distinguished on the basis of independent conversion of AA to TxB2 and PGE2.

Arachidonic Acid↗
PubMed1987

Oxidation of esterified arachidonate by rat liver microsomes.

Rat hepatic microsomal lipids were labeled with [U-14C]arachidonate and were then peroxidized by an NADPH-dependent iron pyrophosphate system. The extent of peroxidation was quantified by malondialdehyde production and arachidonate disappearance. Following peroxidation, the microsomes were centrifuged and the oxidation products were extracted from the supernatant. A linear correlation was found between malondialdehyde production and radioactivity in the supernatant. The pellet was treated with phospholipase A2 to cleave peroxidized products from the phospholipids. Exogenous phospholipase A2 activity was reduced by lipid peroxidation but this was overcome by using a high concentration of the enzyme along with the addition of melittin. The deesterified lipid products from the pellet were extracted and the fragments from the supernatant and the hydrolyzed pellet were separated by reverse-phase HPLC. Several different labeled polar products which coeluted with carbonyl-containing compounds (A285 and hydrazone formation) were found in both the supernatant and the pellet. In addition, many other carbonyl compounds were found which were not arachidonate-derived. The elution pattern of the fragments after 2 and 15 min of peroxidation were qualitatively identical; i.e., no product-precursor relationship was seen. This, along with the observation that peroxidation quickly ceased upon the rapid depletion of NADPH, suggests that propagation did not occur. Finally, the data indicate that cytochrome P-450 is not involved in microsomal lipid peroxidation since product formation is unaffected by the presence of carbon monoxide (80%) and no oxidation of phospholipid arachidonate occurs in the absence of iron.

Animals↗
PubMed1985

Effect of exercise training on antioxidant enzymes and cardiotoxicity of doxorubicin.

The purpose of this study was to correlate the exercise-induced changes of oxidant stress enzymes with possible modification of the response to the putative oxidant stressor doxorubicin. Enzymatic and histological changes were studied in mice placed on a 21-wk swim training program (1 h/day, 5 days/wk) with and without anthracycline administration. Doxorubicin (4 mg/kg) was administered intravenously through a tail vein on 10 separate days over a 7-wk period (twice weekly during weeks 10, 11, 14, 15, and 16). Blood, liver, and heart levels of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GP) were measured following the 9th and 21st wk. Myocardial histomorphological observations were made by light microscopy after 21 wk. Following 9 wk of training swim-trained animals had significantly elevated levels of CAT, SOD, and GP in blood, as well as elevated GP in liver. After 21 wk, trained animals, regardless of drug status, had elevated blood CAT and SOD activity and increased liver CAT and GP. Training also produced increases in blood GP, liver SOD, and heart CAT; however, in conjunction with doxorubicin these changes were not seen. The degree of cardiotoxicity was significantly greater in the sedentary drug-treated animals than in the swim-trained drug-treated animals. The results suggest a correlation between antioxidant enzyme levels in blood and liver and the degree of damage caused by an anthracycline drug. It was concluded that exercise ameliorates severe toxic damage caused by doxorubicin administration, possibly by increasing enzymes that combat free radical damage.

Animals↗
PubMed1984

Prostaglandin response in the exercise conditioned rat.

Thrombin- and collagen-induced thromboxane A2 (TxA2) production were significantly reduced in washed platelets from exercise-trained rats as compared to sedentary controls. No consistant differences were found in the synthesis of prostacyclin (PGI2) from endogenous arachidonate in strips of aorta. Isolated hearts perfused by the "Langendorff" method respond to bolus injections of PGI2 with a decreased perfusion pressure. Hearts from exercised-trained animals were more responsive than those from sedentary animals to the vasodilating effects of PGI2. The increased ratio of PGI2/TxA2 and the increased responsiveness of the coronary vasculature to PGI2 seen in the trained rats may provide protection from cardiovascular disease.

6-Ketoprostaglandin F1 alpha↗
PubMed1977

Fluorescence of Trypan blue in frozen-dried embryos of the rat.

Freeze-drying and fluorescence microscopy techniques were combined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.

Animals↗