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Biomedical subjects

K L Kelly

Publications and source records attributed to K L Kelly.

At least 19 recordsLinked to original sources
PubMed1996

Intracellular pH in adipocytes: effects of free fatty acid diffusion across the plasma membrane, lipolytic agonists, and insulin.

The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH (pH1) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results is a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of Na+/H+ exchange in adipocytes does not prevent the changes in pHi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in pHi occur in adipocytes in response to the passive diffusion of un-ionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in pHi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.

Adipocytes↗
PubMed1995

A phase I study of cisplatin, etoposide, and paclitaxel in small cell lung cancer: a University of Colorado Cancer Center study.

The University of Colorado Cancer Center is conducting a phase I study of the three-drug PET combination of cisplatin, etoposide, and paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in patients with advanced (stage IV or IIIB with pleural effusion) small cell lung cancer. The primary study goal was to define the maximally tolerated doses given on an outpatient basis. Secondary goals were to determine toxicities, response rate, response duration, and survival. Paclitaxel was given as a 3-hour intravenous (IV) infusion prior to cisplatin and etoposide. The starting doses were paclitaxel 135 mg/m2 day I, cisplatin 80 mg/m2 IV day I, and etoposide 50 mg/m2 IV day I, and 100 mg/m2 orally days 2 and 3, every 3 weeks. In the second group, the etoposide was increased to 80 mg/m2 IV day I and 160 mg/m2 orally days 2 and 3. In the third group, paclitaxel was increased to 175 mg/m2 IV day I. Granulocyte colony-stimulating factor was not given on the first cycle, but was given on subsequent cycles if grade 4 neutropenia developed. So far, 13 patients have been entered on the study; all are evaluable for toxicity and nine are evaluable for response. The major toxicity was neutropenia, with no other grade 4 toxicities observed. All patients received the full six cycles of therapy. Thus far, partial responses have been observed in four patients (44%) and complete responses in five patients (56%), for an overall response rate of 100%. This ongoing study has shown that full doses of each of these three active drugs can be administered safely on an outpatient basis. The encouraging early results should lead to a multicenter phase II evaluation of the PET combination.

Ambulatory Care↗
PubMed1994

The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-1. Stimulation by insulin and inhibition by Wortmannin.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a heterodimer composed of an 85-kDa subunit that binds tyrosyl-phosphorylated proteins via its SH2 domains and a 110-kDa catalytic subunit. Expression and mutagenesis experiments have shown that the 110-kDa subunit is a dual specificity kinase that possesses both lipid and serine kinase activities. Except for the 85- and 110-kDa subunits of PI 3-kinase, however, no endogenous substrates for the serine kinase have been identified. The results of the present study show that another target of this kinase is the insulin receptor substrate, IRS-1. Serine phosphorylation of IRS-1 as well as the 85-kDa subunit of PI 3-kinase was demonstrated in immunoprecipitates of PI 3-kinase and IRS-1 isolated from rat adipocytes incubated with insulin. In adipocytes incubated in the absence of insulin, only the serine phosphorylation of p85 was observed in immunoprecipitates of PI 3-kinase. Both the serine and lipid kinase activities of PI 3-kinase were abolished by the fungal metabolite Wortmannin. Wortmannin also partially inhibited the ability of insulin to stimulate glucose transport and inhibit lipolysis in fat cells. These data raise the possibility that the serine kinase activity of PI 3-kinase is involved in insulin signaling. They also suggest that inhibition of the lipid or serine kinase activities of PI 3-kinase could explain the effect of Wortmannin to diminish insulin action.

Adipocytes↗
PubMed1994

Evidence for an ecto-ATPase on the cell wall of Streptococcus sanguis.

Certain strains of viridans streptococci bind platelets, which release ATP from dense granules and then aggregate. By hydrolyzing the released ATP to the platelet agonist, ADP, cell wall-associated ATPase activity of Streptococcus sanguis may amplify the aggregation of platelets. To identify and characterize this ecto-ATPase activity, whole cells were incubated with [14C]-ATP. The cell-free nucleotides were separated by thin-layer chromatography and quantified by liquid scintillation counting. Whole-cell activity showed temperature and pH optima in the physiological range. To isolate a soluble fraction with ATPase activity from the cell wall, whole cells were digested under osmotically stable conditions to produce protoplasts. Protoplasts and cells were separated from soluble cell wall materials by centrifugation. ATPase activity in cell fractions was identified by zymograms of native 8% polyacrylamide gels after electrophoresis. The ecto-ATPase preparation, membrane and cytoplasmic ATPase in lysed protoplasts showed different zymograms and sensitivity to inhibition by DCCD, ouabain vanadate, azide and NEM. In electron micrographs of ultrathin sections of cells of S. sanguis, ATPase activity was localized to the cell wall. Since the pattern of localization to the wall changed with the phase of growth, the ecto-ATPase of S. sanguis may be associated with the development and maintenance of the cell wall.

Adenosine Triphosphatases↗
PubMed1994

Repeated inhibition of cholinesterase by chlorpyrifos in rats: behavioral, neurochemical and pharmacological indices of tolerance.

Previous work from this laboratory showed that daily s.c. injections of the organophosphate diisopropylfluorophosphate caused prolonged inhibition of cholinesterase (ChE) activity in whole blood and brain and downregulation of muscarinic receptors in the central nervous system; these changes were accompanied by progressive, persistent deterioration of working memory and motor function. Further, a single s.c. injection of the organophosphate insecticide chlorpyrifos (O,O',-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothionate, CPF), caused neurochemical changes of the same magnitude and duration, but transient impairment of working memory and motor slowing. In the present study, weekly injections of CPF (0, 15, 30 or 60 mg/kg s.c.) inhibited ChE activity in whole blood of rats by 60% to 90% after 5 weeks; the highest dose also induced tremor, working memory impairment and motor slowing in daily delayed matching-to-position/visual discrimination tests. Reducing the CPF injection frequency to every other week relieved the inhibition of whole blood ChE activity (to 50%-75% of control) and ameliorated all the behavioral deficits. Reinstatement of weekly CPF injections (0, 15, 30, or 45 mg/kg) for 10 weeks inhibited whole blood ChE activity by 75% to 90%. Tremor was not observed during this period; however, motor slowing and working memory impairment persisted throughout the dosing period in all treated groups. Pharmacological evidence for tolerance to the muscarinic effects of CPF was observed on trial completion in the daily delayed matching-to-position/visual discrimination task: CPF-treated rats were supersensitive to scopolamine and subsensitive to pilocarpine. Nicotine reversed the reduction in trial completion associated with CPF. Changes in sensitivity to mecamylamine, d-amphetamine and haloperidol were not observed. Taken together, these studies indicate that inhibition of ChE activity by repeated injection of CPF produces a constellation of behavioral effects not evident after a single CPF treatment, even though both treatment regimens caused prolonged inhibition of ChE activity and downregulation of central muscarinic receptors.

Animals↗
PubMed1993

Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle.

Insulin stimulates the appearance of anti-tyrosine(P)-immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity in adipocytes, predominantly in an intracellular membrane fraction (Kelly, K. L., Ruderman, N. B., and Chen, K. S. (1992) J. Biol. Chem. 267, 3423-3428). Neither the mechanism underlying this activation nor the precise subcellular compartment in which it occurs is known. To address these questions, studies were performed using isolated rat adipocytes and subcellular fractions of these cells. In intact cells, insulin stimulated the rapid appearance of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in 32P-labeled adipocytes without changing the labeling of phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, or phosphatidylinositol 4,5-bisphosphate. This effect was accompanied by the tyrosyl phosphorylation of a 185-kDa protein, tentatively identified as IRS-1, with which PI 3-kinase became associated. The majority of the p85, the regulatory subunit of PI 3-kinase, in untreated adipocytes was present in the cytosol; however, neither the activity of PI 3-kinase nor the total amount of p85 in this fraction was modified in response to insulin. In contrast, insulin increased the association of p85 with IRS-1, the tyrosyl phosphorylation of the IRS-1 associated with p85, and the total activity of PI 3-kinase in the plasma membranes and low density membranes. After insulin treatment, similar amounts of p85 were bound to IRS-1 in the low density and plasma membrane fractions; however, tyrosyl-phosphorylated IRS-1 and PI 3-kinase activity were an order of magnitude greater in the low density membranes. The complex of tyrosyl-phosphorylated IRS-1.p85 that formed in response to insulin was localized to a very low density vesicle subpopulation that could be distinguished from vesicles containing the GLUT-4 glucose transporter and the insulin receptor. These data suggest that the activation of PI 3-kinase by insulin in the adipocyte involves the formation of a complex between IRS-1 and PI 3-kinase in a very low density membrane fraction that is not enriched in GLUT-4 or insulin receptors. They also suggest that PI 3-kinase activation correlates more closely with the extent of tyrosyl phosphorylation of the IRS-1 complexed to PI 3-kinase than it does to the amount of p85 bound to IRS-1.

Adipose Tissue↗
PubMed1993

Methylphenidate and desipramine in hospitalized children: I. Separate and combined effects on cognitive function.

OBJECTIVE: The separate and combined effects of methylphenidate and desipramine on cognitive function were investigated in 16 psychiatrically hospitalized children with primary, secondary, and mixed features of attention-deficit hyperactivity disorder and mood disorder. METHOD: A double-blind, placebo controlled, crossover design was used to investigate drug effects on vigilance, short-term memory, visual problem solving, and higher-order learning. RESULTS: Methylphenidate alone improved vigilance, both drugs positively affected short-term memory and visual problem solving, and combined drugs affected learning of higher-order relationships. CONCLUSIONS: Separate and combined drug effects are related to the specific cognitive domain assessed and have implications for neurotransmitter models of action.

Arousal↗
PubMed1993

Side effects of methylphenidate and desipramine alone and in combination in children.

OBJECTIVE: To investigate side effects of methylphenidate and desipramine alone and in combination in hospitalized children with symptoms of attention-deficit hyperactivity disorder and depression. METHOD: A double-blind placebo controlled crossover design was used to investigate each medication alone and in combination. Side effect ratings and EKGs were done weekly. Pulse and blood pressure were monitored daily. RESULTS: Nausea, dry mouth, and tremor were present in at least twice as many children on combined methylphenidate and desipramine compared with any other condition. Nausea/vomiting, headaches, other aches, refusal of food, and feeling "tired" were significantly more frequent during the combined methylphenidate plus desipramine condition when compared with either methylphenidate alone or with baseline. Significantly higher ventricular heart rate was found on combined methylphenidate plus desipramine compared with desipramine alone, methylphenidate alone, and baseline. Prolonged PR interval and significantly higher heart rate occurred during desipramine alone compared with baseline. CONCLUSIONS: During the several-month duration of the study, there were more frequent side effects during combined methylphenidate plus desipramine treatment than with either medication alone. Clinically, side effects present during combined medication appeared to be similar to and no more serious than those associated with desipramine alone.

Adolescent↗
PubMed1992

In vitro allogeneic cytotoxicity in the solitary urochordate Styela clava.

Hemocytes from the solitary urochordate Styela clava can effect allogeneic cytotoxicity in vitro. Spectrophotometric and microscopic quantification of eosin-y dye exclusion revealed significantly greater frequencies of cell death in allogeneic hemocyte cultures when compared to autogeneic controls. This cytotoxic response was characterized by 1) transient activity such that specific cytotoxicity could be detected for only 4 hours of culture though continued specific killing may have been obscured by spontaneous cell death; 2) a necessity for cellular interaction demonstrated by the elimination of allogeneic cytotoxicity in the absence of cell contact; 3) killing of multiple targets by effector cells due to high levels of response at low allogeneic ratios; 4) insensitivity to altered temperature; 5) increased cytotoxicity in the absence of autologous plasma; 6) an absence of xenogeneic reactivity; 7) the presence of three hierarchical levels (low, intermediate, and high) of response. These data reflect events involved in the recognition of allogeneic cellular determinants resulting in specific cytotoxicity effected by immunocompetent cells. Such in vitro recognition and cytotoxic recognition and cytotoxic reactivity may be responsible for adaptive reactions caused by histoincompatibility in solitary tunicates.

Animals↗
PubMed1992

Phosphatidylinositol-3-kinase in isolated rat adipocytes. Activation by insulin and subcellular distribution.

Insulin increases phosphatidylinositol-3-kinase (PI-3-kinase) activity in Chinese hamster ovary cells transfected with human insulin receptor (Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). The subcellular distribution of PI-3-kinase has not been investigated, and it is unclear if insulin has a stimulatory effect on PI-3-kinase in a nonproliferating target tissue, and, if so, whether this effect is subject to counter-regulation. To address these questions, we studied the effect of insulin on PI-3-kinase activity in isolated rat adipocytes. Activity was measured in plasma membranes, intracellular membranes, and cytosol of control and insulin-treated adipocytes, and in anti-Tyr(P) immunoprecipitates prepared from these fractions and from whole cell lysates. Treatment of adipocytes with insulin (200 nM) caused a half-maximal increase in anti-Tyr(P)-immunoprecipitable PI-3-kinase activity in whole cell lysates within 2 min. This effect was concentration-dependent, and it was sensitive to inhibition by norepinephrine. In insulin-stimulated cells, 75% of anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was found in the low density microsomes. This fraction also exhibited the highest specific activity of PI-3-kinase, and insulin caused a further increase in this activity. Anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was also found in the plasma membranes of insulin-treated cells, but this accounted for only a minor portion of the total and anti-Tyr(P)-immunoprecipitable PI-3-kinase activity. The majority of PI-3-kinase activity (90%) in control cells was cytosolic, but this was not increased in response to insulin nor was it anti-Tyr(P)-immunoprecipitable. These data demonstrate that insulin increases the activity of PI-3-kinase in adipocytes and this effect is subject to inhibition by a physiological antagonist of insulin action. The data also indicate that the effect of insulin to increase PI-3-kinase activity is expressed primarily in the low density intracellular membranes and to a lesser extent in the plasma membranes.

Adipose Tissue↗
PubMed1992

Purification and characterization of a humoral opsonin from the solitary urochordate Styela clava.

1. We have previously identified opsonic activity in the plasma of the solitary urochordate, Styela clava. 2. Here, we report the purification and further characterization of the opsonic molecule. 3. Two purification methods were employed. 4. Gel filtration yielded one strongly opsonic fraction that contained a single, electrophoretically-resolved protein. 5. Opsonic activity was dose-dependent and sensitive to tryptic digestion and heat denaturation. 6. SDS-PAGE and calibrated gel filtration indicated the opsonic protein was a 17.5 kDa monomer while isoelectrofocusing indicated a single pI of 7.0. 7. In an alternative procedure, a similar opsonic activity and protein were isolated by affinity purification using whole yeast cells.

Animals↗
PubMed1992

The effects of methylphenidate and lithium on attention and activity level.

Seven psychiatrically hospitalized prepubertal children were treated with methylphenidate, placebo, lithium carbonate alone and in combination with methylphenidate. Children met DSM-III-R criteria for both disruptive behavior disorder and bipolar or major depressive disorder by structured interview and consensus diagnosis. Parents, children, teachers, staff, and raters were blind to medication condition. Children were rated weekly on measures of attention and activity to determine whether medications alone or in combination produced a differential effect on these variables. The nosological and practical implications of the results are discussed.

Arousal↗
PubMed1992

Lithium in hospitalized children at 4 and 8 weeks: mood, behavior and cognitive effects.

Eleven psychiatrically hospitalized children were treated with lithium carbonate for a minimum of 8 weeks. Based on literature review, their psychopathology, diagnoses and family history suggested they would be positive lithium responders. Weekly ratings in behavioral domains showed children's improvement in areas of self control, aggression and irritability, more obvious at 8 than 4 weeks. However, of seven children studied with double blind crossover, improvement behaviorally and cognitively was maintained on placebo. Only three of 11 children improved enough to be discharged on lithium. Implications of lithium's anti-aggressive and anti-manic effects in children are discussed.

Affect↗
PubMed1989

Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action.

It has been proposed that an elevation in cytosolic free Ca2+ may play a role in either mediating or antagonizing the ability of insulin to stimulate glucose uptake in adipocytes. This question has been addressed in the present studies using isolated fura-2-loaded rat adipocytes stimulated with a variety of agonists. The effects of insulin, oxytocin, norepinephrine, ATP, and ionomycin on cytosolic free Ca2+ levels were assessed and compared with their effects on transport-limited glucose oxidation. Oxytocin and ionomycin at concentrations which caused 3-5-fold increases in cytosolic Ca2+, by releasing Ca2+ from internal stores, had no effect on insulin-stimulated glucose oxidation. ATP and norepinephrine which caused more modest increases in Ca2+, by mechanisms at least partially dependent on external stores, inhibited insulin-stimulated glucose oxidation. Insulin had no effect on basal Ca2+ levels nor did it modulate the Ca2+ elevation caused by other agonists. These data suggest that insulin-stimulated glucose transport is not associated with an increase in cytosolic Ca2+. In addition, although there appears to be a correlation between inhibition of insulin-stimulated glucose transport and the effect of certain agonists to promote Ca2+ influx, there is not a general obligatory relationship between an elevation in cytosolic Ca2+ and antagonism of this insulin action.

Adenosine Triphosphate↗
PubMed1989

Attention deficit disorder with hyperactivity and methylphenidate: the effects of dose and mastery level on children's learning performance.

This investigation examined the relationship between methylphenidate (MPH) and the learning and recall of paired associations by children with attention deficit disorder with hyperactivity (ADDH). Forty-five children with ADDH were randomly assigned to one of three groups (novel, partial mastery, and mastery learning) that varied in the amount of previous learning of paired associations and participated in a double-blind, placebo-control, repeated-measures-across-dose (crossover) design. Each child received four doses of MPH (5 mg, 10 mg, 15 mg, and 20 mg) and a placebo in a random, counterbalanced sequence. The results indicated that both the rate of acquisition and accuracy in learning paired associations were significantly, but differentially, affected by MPH dose and the degree of learning mastery. The implications of these results for psychopharmacological research and the monitoring of psychostimulant effects on children's learning performance in academic settings are discussed.

Association Learning↗