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Biomedical subjects

C Ramachandran

Publications and source records attributed to C Ramachandran.

At least 19 recordsLinked to original sources
PubMed1995

Direct determination of the sequence recognition requirements of the SH2 domains of SH-PTP2.

SH-PTP2 is a widely-expressed protein tyrosine phosphatase with two tandem SH2 (src homology 2) domains and a C-terminal catalytic domain. Glutathione S-transferase fusions of the SH2 domains alone and of a catalytically inactive full-length mutant were made, and binding assays were developed using the purified fusion proteins to directly determine what residues are involved in the recognition of binding targets by the SH2 domains. The binding kinetics of the SH2 domains to a phosphotyrosyl-containing peptide of the sequence surrounding Tyr1009 of the platelet-derived growth factor receptor (PDGFR) beta subunit [DTSSVL(pY)TAVQPN] were determined by surface plasmon resonance, confirming that this is a high-affinity binding ligand. Using various N- and C-terminal truncations of this peptide as competitors in the binding assays, the minimum peptide that served as a high-affinity binding ligand was found to be VL(pY)TAV. Systematic Ala substitutions of this peptide indicated that in addition to the phosphotyrosine (pY), the critical residues for recognition and binding are at pY + 1 and pY + 3 as previously reported, and notably at pY-2 as well. Binding competition results with these and other PDGFR, IRP, and IRS-1 peptides suggested some general rules for sequence recognition by the SH2 domains of SH-PTP2. Peptides that bind to the SH2 domains in the binding assays were also found to stimulate the phosphatase activity of SH-PTP2.

Amino Acid Sequence↗
PubMed1995

Topical delivery of growth hormone releasing peptide using liposomal systems: an in vitro study using hairless mouse skin.

The results of this study clearly demonstrates the utility of novel non-ionic liposomal systems in facilitating transfer of GHRP-6 into and across deeper strata of skin following topical application. These findings indicate that it may be possible to deliver therapeutic doses of a wide variety of peptides to local skin tissue via topical application. The results also suggest the possibility of controlled enhancement of skin penetration or metered polypeptide deposition through appropriate choice of liposomal lipid components. The pronounced enhancement of GHRP-6 and mannitol transport from emulsions containing the nonionic lipids suggests a promising delivery system for hydrophilic drugs in general.

Administration, Cutaneous↗
PubMed1995

Transfollicular drug delivery.

The hair follicle, hair shaft, and sebaceous gland collectively form what is recognized as the pilosebaceous unit. This complex, three-dimensional structure within the skin possesses a unique biochemistry, metabolism and immunology. Recent studies have focused on the hair follicle as a potential pathway for both localized and systemic drug delivery. Greater understanding of the structure and function of the hair follicle may facilitate rational design of drug formulations to target follicular delivery. Targeted drug delivery may enhance current therapeutic approaches to treating diseases of follicular origin. Presented here is a review of follicular drug delivery and a discussion of the feasibility of the pilosebaceous unit as a target site.

Administration, Topical↗
PubMed1995

Influence of nonionic liposomal composition on topical delivery of peptide drugs into pilosebaceous units: an in vivo study using the hamster ear model.

PURPOSE: The purpose of this study was to test the hypothesis that nonionic liposomes facilitate the topical delivery of peptide drugs into pilosebaceous units. METHODS: The hamster ear was used as a model for human pilosebaceous units. The deposition of a hydrophilic protein, alpha-interferon (alpha-IFN), into pilosebaceous units and other strata of the hamster ear 12 hours after topical in vivo application of three nonionic liposomal formulations, one composed of glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether (Non-1), the second composed of glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether (Non-2) and the third composed of polyoxyethylene-10-stearyl ether/cholesterol (Non-3), a phospholipid-based liposomal formulation (PC) and an aqueous control solution (AQ) was determined. We also determined the deposition of a hydrophobic peptide, cyclosporin-A (CsA), into pilosebaceous units and other strata of the hamster ear after topical in vivo application of these liposomal formulations and a hydroalcoholic control solution (HA). RESULTS: The deposition of alpha-IFN into the pilosebaceous units was in the order: Non-1 >> PC > Non-2 > Non-3 = AQ. The deposition of CsA into the pilosebaceous units was in the order: Non-1 >> HA > PC > Non-2 = Non-3. CONCLUSIONS: Despite differences in the hydrophobicities and size of the drug molecules, deposition into the various ear strata was significantly enhanced by the Non-1 liposomal system.

Administration, Topical↗
PubMed1995

In vitro and in vivo biotransformations of the naphthalenic lignan lactone 5-lipoxygenase inhibitor, L-702,539.

It has been reported previously that the tetrahydropyranyl naphthtalenic lignan lactone L-702,539 is a potent nonredox, 5-lipoxygenase inhibitor that has the advantage that it can be dosed either as the lactone or as the corresponding nonactive hydroxy acid L-702,618 (opened lactone). Studies with hepatic microsomes from the rat, rhesus monkey, and human were undertaken in a phosphate buffer and suggested that the closure of the hydroxy acid L-702,618 to the lactone L-702,539 was an enzymatic process. The incubation of L-702,539 under oxidative conditions with these specific hepatic microsomes resulted in the formation of three significant metabolites (> 0.4 nmol/mg protein/hr) as determined by HPLC with UV detection. These metabolites were isolated from large microsomal incubations and were characterized by MS and NMR spectroscopy. Data showed that the lactone and tetrahydropyran portions of the molecule were both susceptible to hydroxylation, and the hydroxylated tetrahydropyran was further oxidized to the hydroxy acid. Analysis of plasma samples obtained from rat and rhesus monkeys following L-702,618 administration indicated that the in vivo metabolic pathway was similar to the one observed in vitro using hepatic microsomes. Studies conducted with microsomes from genetically engineered human cell lines expressing individual cytochrome P450s indicated that the isozyme responsible for the metabolism at the tetrahydropyran ring, was P4503A4. These findings were supported by studies conducted in human microsomes using an inhibitory P4503A4 antibody and troleandomycin, which is a potent P4503A inhibitor.

Animals↗
PubMed1994

Liposomes: a novel topical delivery system for pharmaceutical and cosmetic applications.

This review will highlight work done in our laboratories evaluating topical liposomal delivery in a wide variety of animal models and human skin using both in vivo and in vitro techniques. The mechanism by which liposomes facilitate deposition of drugs into the skin and some potential applications of topically applied liposomes will be discussed. Particular emphasis will be placed on the development of analytical techniques that allow the quantification of drug levels in the various skin strata and in pilosebaceous structures. Appropriate models of skin representing the two cases, as well as those wherein both routes are available for skin deposition, were examined using a wide variety of liposomal preparations containing radiolabeled and fluorescent molecules as marker compounds.

Animals↗
PubMed1993

Inhibition of the activity of protein tyrosine phosphate 1C by its SH2 domains.

Full-length protein tyrosine phosphatase 1C (PTP1C), the catalytic domain of PTP1C (delta PTP1C), and the N-terminal SH2 domain truncated PTP1C (delta NPTP1C) were overexpressed in Escherichia coli and purified to near homogeneity. Various phosphorylated states of the synthetic phosphotyrosyl peptide TRDIYETDYYRK (IRP), corresponding to the major insulin receptor autophosphorylation sites, were used as substrates for the PTPs. There was no indication for selective dephosphorylation of any of the three phosphotyrosyl residues from the triphosphotyrosyl IRP. Kinetic studies were carried out using all seven different phosphotyrosyl IRPs. Saturation kinetics were observed for PTP1C using the triphosphotyrosyl IRP only. In contrast, for delta PTP1C, saturation was achieved for all seven phosphotyrosyl IRPs. The best substrate for delta PTP1C was the triphosphotyrosyl IRP possessing a Km of approximately 1.6 microM, about 3-4-fold lower than either the mono- or diphosphotyrosyl IRPs. However, in contrast to delta PTP1C, PTP1C had a 22-fold lower affinity for triphosphotyrosyl IRP. Furthermore, deletion of a single N-terminal SH2 domain increased the affinity of the enzyme for the triphosphotyrosyl IRP to a value similar to that obtained with delta PTP1C. The pH optima for all three enzyme constructs were very similar and could not account for the observed change in substrate affinity between the three enzymes. These results suggest that the SH2 domain of PTP1C exerts an inhibitory effect on its PTP activity.

Amino Acid Sequence↗
PubMed1992

Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.

Adenosine Triphosphate↗
PubMed1992

Sequential dephosphorylation of a multiply phosphorylated insulin receptor peptide by protein tyrosine phosphatases.

The question of whether protein tyrosine phosphatases (PTPases) dephosphorylate a multiply phosphorylated peptide in a random or ordered manner was investigated using the synthetic triphosphotyrosyl peptide TRDIY(P)ETDY(P)Y(P)RK, corresponding to the major sites of autophosphorylation of the insulin receptor, as a substrate for four purified PTPases. All four enzymes dephosphorylated the triphospho peptide to produce diphospho, monophospho, and nonphosphorylated forms. Partially dephosphorylated peptides were separated by reverse-phase HPLC, and the di- and monophospho peptides were collected and analyzed by solid-phase sequencing to determine the order of dephosphorylation of the three sites by each of the PTPases. The quantitative analysis of the signals for derivatives of tyrosine and phosphotyrosine generated at positions 5, 9, and 10 of the peptide showed that the low molecular weight human placental PTPase 1B preferentially dephosphorylated the two phosphotyrosines at positions 9 and 10 whereas the integral membrane enzyme CD45 (from human spleen) and the bacterially expressed rat LAR preferentially dephosphorylated the phosphotyrosine at position 5. A second low molecular weight enzyme, termed TCPTPase, did not display any specificity for a particular phosphotyrosyl residue. These results demonstrate that different PTPases exhibit a characteristic pattern of dephosphorylation of the triphospho peptide model substrate, raising the possibility that features in the primary structure surrounding the dephosphorylation site may contribute to substrate specificity.

Amino Acid Sequence↗
PubMed1992

Bulk organic solvent-water systems as a possible model to predict alkyl p-aminobenzoate partitioning in liposomes.

This study compares the bilayer-water distribution coefficients of a homologous series of n-alkyl p-aminobenzoates in liposomes with their respective distribution coefficients in octanol-water, oleyl alcohol-water, and hexane-water systems. The data indicate that the bilayer-water distribution coefficient is quite sensitive to changes in solute structure and to the structural organization of the bilayer and that octanol, oleyl alcohol, and hexane are able to reflect the partitioning changes that occur in the liposomal bilayer with respect to increasing the alkyl chain length of n-alkyl p-aminobenzoates. For this particular homologous series, the hexane-water system tended to underestimate the bilayer-water distribution coefficients, whereas octanol-water and oleyl alcohol-water systems overestimated solute partitioning into the bilayer. The similarity of the lipid environment, with respect to solute partitioning, in the organic solvent system and that in liposomes can be ascertained by using the Collander relationship.

4-Aminobenzoic Acid↗
PubMed1992

Topical delivery of liposomally encapsulated gamma-interferon.

The extent of uptake of gamma interferon (gamma-IFN) in various strata of hairless mouse, human and hamster skin upon application of a liposomal formulation and an aqueous solution were determined by in vitro diffusion cell experiments. For each of the animal species studied, 70-80% of the liposomally entrapped IFN was deposited onto or penetrated into the skin as determined 24 h after in vitro application. However, a significant fraction of this total amount (approximately 0.25-0.30) is either adsorbed to or associated with the stratum corneum. The drug content found in the deeper skin strata, where the receptor sites reside, suggests that drug deposition is strongly influenced by the skin species tested. The percent of applied drug found in this strata 24 h after application followed the order: hamster (6.1) much greater than human (0.9) greater than hairless mouse (0.3), although the amounts of drug in the total skin of each species tested were approximately the same. This indicates that the deposition of drug into the living epidermis and/or dermis cannot be predicted by determination of the amount of drug in the total skin. The amounts in the deeper skin strata were also in the order of increasing number of follicles/hair in the skin species, suggesting that the transfollicular route is an important pathway for liposomal topical therapeutics.

Administration, Topical↗
PubMed1992

Topical delivery enhancement with multilamellar liposomes into pilosebaceous units: I. In vitro evaluation using fluorescent techniques with the hamster ear model.

Evidence suggesting liposomal delivery into the pilosebaceous unit of the male Syrian hamster ear membrane was found using two fluorescent techniques, quantitative fluorescence microscopy (QFM), and a scraping method where the various tissue strata of treated skin are analyzed using fluorescence spectrophotometry. Whole ears were mounted on Franz diffusion cells and treated for 24 h with 40 microliters of the following test formulations, each containing approximately 100 micrograms/ml carboxyfluorescein (CF): i) multilamellar phosphatidylcholine: cholesterol: phosphatidylserine liposomes; ii) HEPES buffer (pH, 7.4); iii) 5% propylene glycol; iv) 10% ethanol; v) 0.05% sodium lauryl sulfate; and vi) a suspension of the same lipids used to form the liposomes that were not processed so as to produce a bilayer configuration. Topical application of the liposomally based formulation resulted in a significantly higher accumulation of CF in the pilosebaceous units than the application of any of the other non-liposomal formulations. There was excellent correlation between the two analytical methods used to determine CF deposition into the sebaceous glands.

Administration, Topical↗
PubMed1991

Murine progesterone receptor exists predominantly as the 83-kilodalton 'A' form.

Progesterone receptors (PgR) are known to exist in two molecular forms commonly designated as 'A' and 'B' forms, and the relative ratio of these two forms has been shown to vary among species. Although the rodent systems were some of the earliest experimental systems used to examine the regulation of PgR, as yet very little is known concerning the molecular composition of PgR in this species. Accordingly, to define the relative ratio of 'A' and 'B' forms in murine PgR, we have analyzed tissue extracts from normal, ovariectomized, and estradiol treated animals by photoaffinity labeling and immunoblotting techniques using a variety of anti-PgR antibodies. Under all experimental conditions, two forms of PgR with approximate molecular weights of 115 kDa ('B' form) and 83 kDa ('A' form) were found. In all tissues examined, the 83 kDa 'A' form was predominant, and this was independent of the hormonal status of the animal and different buffers used to prepare tissue extracts. In uterus the ratio of 'A' to 'B' was 3:1, in vagina it was 2:1, and in mammary glands it more closely resembled the uterus. This leads us to conclude that murine PgR exists predominantly as the 83-kDa 'A' form which may represent a general characteristic of rodent PgR. In this species there may also be some tissue specificity with regard to the absolute ratio of the two forms of PgR.

Affinity Labels↗
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↗