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Biomedical subjects

C Ramachandran

Publications and source records attributed to C Ramachandran.

At least 73 records · Page 4Linked to original sources
PubMed1991

Topical application of liposomally entrapped cyclosporin evaluated by in vitro diffusion studies with human skin.

The kinetics and extent of uptake of cyclosporin (CSA) in various strata of human cadaver skin upon topical application of several CSA formulations were determined by in vitro diffusion cell experiments. The CSA formulations tested included an oil-in-water emulsion and four liposomal systems. The accumulation of CSA in the stratum corneum at 24 h is in the order: 'skin lipid' multilamellar liposomes (MLV) greater than phospholipid MLV approximately 'skin lipid' large unilamellar liposomes (LUV) greater than phospholipid LUV much much greater than emulsion. The total amount of drug in the deeper stratum corneum and deeper skin strata at 24 h is in the order: phospholipid MLV greater than 'skin lipid' MLV greater than phospholipid LUV greater than 'skin lipid' LUV greater than emulsion. Whereas 'skin lipid' liposomes were more effective than phospholipid-based liposomes in depositing drug in deeper skin strata for rodent species (mouse and guinea pig), the opposite effect was observed for human cadaver skin. More importantly, all the liposomal formulations tested were far more effective than the emulsion formulation in depositing CSA into the skin.

Administration, Topical↗
PubMed1990

Topical delivery of liposomally encapsulated interferon evaluated by in vitro diffusion studies.

The topical delivery of several liposomal interferon formulations was evaluated by in vitro diffusion experiments in an effort to understand the effects of liposomal composition and method of preparation on the deposition of interferon into the stratum corneum and deeper strata of the skin. Application of liposomes prepared from lipids with a composition similar to that of the stratum corneum resulted in almost twice the amount of interferon being deposited in the deeper skin layers than did application of liposomes prepared from phospholipids. Topical application of "skin lipid" liposomes prepared by the dehydration-rehydration method was twice as effective as was topical application of liposomes prepared by the reverse-phase evaporation method with respect to their ability to deposit interferon into the skin strata where the basal cell layers reside. These results are consistent with the effects of liposomal composition and method of preparation on the ability of the formulation to reduce lesion scores in the cutaneous herpes simplex virus type 1 guinea pig model.

Animals↗
PubMed1990

Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase.

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Topical delivery of ciclosporin: evaluation of various formulations using in vitro diffusion studies in hairless mouse skin.

The kinetics and extent of uptake of ciclosporin in various strata of hairless mouse skin upon topical application of several ciclosporin formulations were determined by in vitro diffusion cell experiments. The ciclosporin formulations tested included a hydroalcoholic solution, an oil-in-water emulsion and two liposomal systems. The accumulation of drug in stratum corneum is in the order: 'skin lipid' liposomes greater than 'phospholipid' liposomes greater than emulsion greater than hydroalcoholic solution. The total combined amount of drug in the deeper skin strata and the receiver compartment followed the order: hydroalcoholic solution much greater than 'phospholipid' liposomes greater than 'skin lipid' liposomes greater than emulsion. The results suggest that topically applied liposomes, particularly those prepared from lipid mixtures having compositions similar to the stratum corneum, may provide sustained, enhanced levels of ciclosporin in the stratum corneum (the reservoir) while minimizing high levels in strata associated with blood and lymph supplies.

Administration, Topical↗
PubMed1989

Developmental regulation of murine mammary-gland 90 kDa heat-shock proteins.

We have examined the regulation of murine mammary-gland 90 kDa heat-shock protein (hsp-90) as a function of normal development and differentiation. We find that both hsp-90 and amounts of its mRNA are modulated during development and differentiation, with the highest concentrations of mRNA and protein being present in tissues from pregnant and lactating animals respectively. Metabolic labelling experiments with [35S]methionine reveal that the rate of synthesis of hsp-90 also varies among tissues from various developmental states and correlates with the relative hsp-90 mRNA content. These data also suggest that the highest concentration of hsp-90 found in lactating mammary tissues may be due to a greater stability of this protein in this developmental state. The possible significance of the developmental modulation of mammary hsp-90 to mammary steroid-receptor properties is discussed.

Animals↗
PubMed1989

The renal Na+/Ca2+ exchange system is located exclusively in the distal tubule.

The movement of Ca2+ across the basolateral plasma membrane was determined in purified preparations of this membrane isolated from rabbit proximal and distal convoluted tubules. The ATP-dependent Ca2+ uptake was present in basolateral membranes from both these tubular segments, but the activity was higher in the distal tubules. A very active Na+/Ca2+ exchange system was also demonstrated in the distal-tubular membranes, but in proximal-tubular membranes this exchange system was not demonstrable. The presence of Na+ outside the vesicles gradually inhibited the ATP-dependent Ca2+ uptake in the distal-tubular-membrane preparations, but remained without effect in those from the proximal tubules. The activity of the Na+/Ca2+ exchange system in the distal-tubular membranes was a function of the imposed Na+ gradient. These results suggest that the major differences in the characteristics of Ca2+ transport in the proximal and in the distal tubules are due to the high activity of a Na+/Ca2+ exchange system in the distal tubule and its virtual absence in the proximal tubule.

Animals↗
PubMed1989

Influence of insulin on phosphate uptake by brush border membranes from human placenta.

Regulation of phosphate transport by insulin was investigated in brush border membranes from human placenta at term. At 22 degrees C, a 45 min incubation of the total tissue with 10(-6) M insulin significantly decreased both the initial rate and the peak of sodium-dependent phosphate uptake by the corresponding brush border membranes. In contrast, Na+ transport was not influenced by the hormone. Increasing the insulin concentration from 0 to 10(-5) M resulted in a dose-dependent inhibition of phosphate uptake with half-maximal effect at 1.1 x 10(-9) M. The hormone decreased PO4 transport by decreasing the affinity of the carrier for the substrate (Km = 0.180 +/- 0.010 mM and 0.215 +/- 0.015 mM in absence and presence of 10(-6) M insulin respectively, P less than 0.05). The inhibitory effect of insulin required the presence of Mn2+ whereas neither Mn2+ nor insulin alone had any influence on PO4 uptake. It is therefore assumed that receptor phosphorylation, which needs the presence of Mn2+, is an intermediate step of insulin action on PO4 uptake by the subsequently isolated brush border membranes. In contrast, insulin had no effect on PO4 uptake when the membranes were directly incubated with the hormone prior to the transport measurement, suggesting that an intracellular messenger is needed for the inhibitory effect. This messenger is not cAMP since insulin at 10(-6) M concentration has no effect on cAMP content of the total placental tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP↗
PubMed1989

Topical delivery of liposomally encapsulated interferon evaluated in a cutaneous herpes guinea pig model.

The topical delivery of liposomally encapsulated interferon was evaluated in the cutaneous herpes simplex virus guinea pig model. Application of liposomally entrapped interferon caused a reduction of lesion scores, whereas application of interferon formulated as a solution or as an emulsion was ineffective. The method of liposomal preparation rather than the lipid composition of the bilayers appeared to be the most important factor for reducing lesion scores. Only liposomes prepared by the dehydration-rehydration method were effective. This finding implied that the dehydration and subsequent rehydration of the liposomes facilitate partitioning of the interferon into liposomal bilayers, where the drug is positioned for transfer into the lipid compartment of the stratum corneum. Liposomes do not appear to function as permeation enhancers but seem to provide the needed physicochemical environment for transfer of interferon into the skin.

Administration, Topical↗
PubMed1988

Estrogenic regulation of uterine 90-kilodalton heat shock protein.

Recently two lines of evidence have implicated that cellular heat shock proteins (hsp) may play a role in steroid hormonal regulation of target tissues. One is the demonstration that cellular 90K hsp (hsp-90) can complex with steroid receptors in vitro and inhibit their ability to interact with DNA, and second, the demonstration that in avian oviduct sex steroids can regulate the synthesis of hsp-108. As yet, there is no report that sex steroids can regulate hsp-90 synthesis, especially in mammalian tissues. In these studies we have examined the estrogenic regulation of murine uterine hsp-90. We report that ovariectomy reduces the uterine concentration of hsp-90, and estradiol causes a time-dependent increase in uterine hsp-90 as early as 4 h after steroid administration, reaching a maximum increase of 4-fold between 18-24 h. The effect is specific to estrogens and not elicited by other steroid hormones. It is also target tissue specific, such that it is seen with uterus and vagina and does not occur in nontarget tissue for estradiol, such as spleen. The possible physiological significance of estrogenic stimulation of uterine hsp-90 has been discussed.

Androgens↗
PubMed1987

The interrelationship between cAMP-dependent alpha and beta subunit phosphorylation in the regulation of phosphorylase kinase activity. Studies using subunit specific phosphatases.

This study addresses the function of multisite phosphorylation of phosphorylase kinase catalyzed by the cAMP-dependent protein kinase. Using subunit specific protein phosphatases (the polycation-stimulated and ATP-, Mg2+-dependent enzymes), we show that the degree of phosphorylation of both the alpha and beta subunits modulates phosphorylase kinase activity. beta subunit phosphorylation is essential for activation and, independent of the degree of alpha subunit phosphorylation, enzyme fully dephosphorylated in the beta subunit is completely inactivated. alpha Subunit phosphorylation does, however, also regulate activity, and enzyme fully or partially phosphorylated in the beta subunit is inactivated as a consequence of alpha subunit dephosphorylation. The extent of inactivation caused by alpha subunit dephosphorylation is linearly dependent on the phosphorylation state of the beta subunit. Three peptide sites on the alpha subunit are phosphorylated by the cAMP-dependent protein kinase; the site primarily affecting activity is the one that is initially phosphorylated. These data provide evidence that subunit interrelationships play an important role in the regulation of phosphorylase kinase by multisite phosphorylation.

Adenosine Triphosphate↗
PubMed1987

Subunit phosphorylation and activation of phosphorylase kinase in perfused rat hearts.

The potential correlations between phosphorylase kinase subunit phosphorylation and activation have been examined using 32P-perfused rat hearts exposed to a variety of hormonal stimuli. Phosphate incorporation was measured after isolation of the enzyme by immunoprecipitation from heart extracts. Time courses of catecholamine or glucagon treatment produced a rapid rise in both the activity and the beta subunit phosphorylation of the enzyme, and a slightly slower increase in alpha' subunit phosphorylation. For short durations of catecholamine stimulation, the ratio of phosphate in the alpha' versus beta subunit was dependent upon hormone dose. After removal of hormone, both inactivation and alpha' subunit dephosphorylation were fairly slow, while the beta subunit was dephosphorylated more rapidly. For all of the above conditions, activation correlated with both alpha' and beta subunit phosphorylation. The maximum level of phosphate incorporation observed in response to hormonal stimulation is estimated to be approximately 1.3-1.7 mol of [32P]phosphate/mol of (alpha' beta gamma delta)4, divided about equally between the alpha' and beta subunits. When hearts were treated with hormone either in the absence of added calcium or in the presence of a calcium channel blocker, the time courses of subunit phosphorylation and activation were similar to those seen with standard perfusion conditions, suggesting that if any Ca2+-dependent autophosphorylation of phosphorylase kinase were occurring it does not make a major contribution to the observed hormonal responses. The complicated relationships observed here between phosphorylase kinase subunit phosphorylation and activation for the most part provide physiological affirmation of the patterns observed in vitro, but they also show some possible differences of potential interest.

Animals↗
PubMed1984

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase.

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1983

Hormonal regulation of the phosphorylation of glycogen synthase in perfused rat heart. Effects of insulin, catecholamines, and glucagon.

32P-labeled perfused rat hearts were used to study the hormonal regulation of glycogen synthase. Following equilibration of perfused hearts with inorganic [32P]phosphate for 30 min, there was an incorporation of approximately 200 pmol of [32P]phosphate/unit of enzyme activity that arose from an exchange of [32P] phosphate with the endogenous [31P]phosphate. Maximum insulin-induced activation (10 milliunits/ml for 5 min), which promoted an I/D activity ratio change from 25% I to 40% I, was associated with a 22% decrease in phosphate content of the enzyme. With hearts from alloxan-induced diabetic animals, there was a 17% higher level of phosphate incorporation and a 4-fold decrease in % I glycogen synthase activity compared to normal animals, but with the diabetic tissue, insulin added to the perfusate had no effect on either the phosphate content or the activity ratio of the enzyme. In perfused hearts from normal animals, DL-isoproterenol and glucagon caused an increase in glycogen synthase phosphorylation of 85-100 pmol/unit of enzyme activity, while L-phenylephrine increased the phosphate content by only 20-35 pmol, but all three hormones caused the same degree of inactivation. The increase in cardiac glycogen synthase phosphorylation induced by DL-isoproterenol, glucagon, and L-phenylephrine was identical, with or without insulin pretreatment; this despite the fact that these three hormones promoted a 3- to 4-fold larger decrease in the enzyme activity ratio with the insulin-treated tissue. In perfused diabetic hearts, DL-isoproterenol, glucagon, and L-phenylephrine caused increased phosphorylation of glycogen synthase without affecting the albeit already low activity ratio of the enzyme. These results show that in the intact perfused heart the same degree of glycogen synthase inactivation can occur as a consequence of differing degrees of phosphorylation, presumably due to phosphorylation at different sites promoted by different second messengers. Conversely, the data indicate that the same extent of phosphorylation, as stimulated by the same second messenger, can inactivate glycogen synthase by different amounts depending upon the prior phosphorylation state of other sites in the protein.

Animals↗
PubMed1983

Regulation of cardiac glycogen synthase.

In perfused rat hearts insulin can activate, and catecholamines can inactivate, glycogen synthase (EC 2.4.1.11); the magnitude of each hormonal response is magnified if tissue glycogen levels are depleted. Both beta-adrenergic and alpha-adrenergic agonists inactivate insulin-stimulated and basal glycogen synthase, with each promoting the same extent of inactivation in both circumstances. In this system beta-adrenergic agonists act via cyclic AMP (cAMP), and alpha-adrenergic agonists via Ca2+, whereas insulin action appears to be independent of either cAMP or Ca2+. The action on cardiac glycogen synthase by the physiological catecholamine epinephrine is apparently mediated by the concomitant interaction with both alpha and beta receptors; interaction with each is mediated by their separate second messenger systems, which combine to produce the end physiological response.

Animals↗
PubMed1982

Cyclic AMP-dependent and cyclic AMP-independent antagonism of insulin activation of cardiac glycogen synthase.

The hormonal regulation of glycogen synthase has been studied with isolated perfused hearts that were depleted of 85% of their endogenous glycogen. Glycogen depletion alone promoted a 3-fold activation of glycogen synthase and magnified by 3-fold the response to insulin. Glycogen depletion also facilitated the detection of epinephrine-promoted glycogen synthase inactivation. Hormonal effects on glycogen synthase have been correlated with changes in phosphorylase, phosphorylase kinase, and tissue cAMP levels. Insulin activation of glycogen synthase was observed within 90 s of hormone addition and was maximal by 4 min. A half-maximum effect was obtained at an insulin concentration of 100 microunits/ml. Insulin-dependent activation is reversed by beta-adrenergic agonists, alpha-adrenergic agonists, and glucagon. Each promote the same degree of inactivation and the maximum extent of inactivation produced by each is independent of whether or not the tissue has been stimulated with insulin. beta-Adrenergic agonists and glucagon act via cAMP, alpha-agonists most likely act via intracellular Ca2+ translocation, and insulin action would appear to be independent of either cAMP or Ca2+. The action of epinephrine on cardiac glycogen synthase is mediated by interaction with both alpha- and beta-receptors. As indicated by dose-response curves, receptor occupancy of each occurs to an almost equal extent at suboptimal epinephrine concentrations. Regulation of cardiac glycogen synthase by epinephrine thus is mediated by two second messenger systems which converge to produce the end physiological response.

Animals↗