Biomedical subjects

C Ramachandran

Publications and source records attributed to C Ramachandran.

At least 37 records · Page 2Linked to original sources

PubMed1999

Differential sensitivity of human mammary epithelial and breast carcinoma cell lines to curcumin.

Curcumin has anti-inflammatory, antiproliferative, and antitumor effects. To understand the chemopreventive mechanism of curcumin against human malignancies, the cellular and molecular changes induced by this agent in human mammary epithelial (MCF-10A) and breast carcinoma (MCF- 7/TH) cell lines were investigated. The human multidrug- resistant breast cancer cell line was 3.5 fold more sensitive to curcumin than the mammary epithelial cell line. Even though both cell lines accumulated a similar amount of curcumin, a significantly higher percentage of apoptotic cells was induced in breast cancer cells compared to a very low percentage of apoptosis in mammary epithelial cells. Incubation of breast cancer cells with 20 and 40 microM curcumin for 24 h induced G2 block and sub-G0/G1 cell population, respectively. Curcumin treatment caused a reduction in the expression of Ki67, PCNA, and p53 mRNAs in breast cancer cells. The human mammary epithelial cell line showed a down-regulation of p21 mRNA and an up-regulation of Bax mRNA expression with curcumin treatment. The results suggest that apoptosis is involved in the curcumin-induced inhibition of tumor cell growth, and genes associated with cell proliferation and apoptosis may be playing a role in the chemopreventive action of curcumin.

Algorithms↗

PubMed1999

3,6-Fluorescein Diphosphate: A Sensitive Fluorogenic and Chromogenic Substrate for Protein Tyrosine Phosphatases*

A highly sensitive and continuous protein tyrosine phosphatase (PTPase) assay using 3,6-fluorescein diphosphate (FDP) is described. Leukocyte phosphatase CD45 (leukocyte common antigen), protein tyrosine phosphatase-1B, and leukocyte common antigen-related protein LAR preferentially hydrolyze FDP to fluorescein monophosphate (FMP) with V(max) and K(m) values comparable with those of phosphotyrosine peptide substrates. Further hydrolysis of FMP to fluorescein was less efficient because of increased K(m) values compared with those of FDP. FMP absorbs strongly at 445 nm and fluoresces intensely near 515 nm, both of which are insensitive to pH perturbations above pH 6. Its high catalytic efficiency, coupled with the highly sensitive dual detection in the visible wavelength region and wider pH operating range, make FDP the substrate of choice for PTPase inhibitor screening in HTS format and assay miniaturization.

Journal Article↗

PubMed1998

Phorbol ester-induced P-glycoprotein phosphorylation and functionality in the HTB-123 human breast cancer cell line.

The discordance between P-glycoprotein (P-gp) expression and functionality [as measured by the efflux of doxorubicin (DOX)] was analyzed in a DOX-sensitive human breast cancer cell line (HTB-123) with high reactivity against four P-gp specific monoclonal antibodies (C219, MRK-16, UIC2, and 4E3). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analyses confirmed the overexpression of MDR1 mRNA and P-gp in this cell line. However, incubation of cells with efflux blockers, verapamil (VPL) or dipyridamole (DPD), did not enhance cellular (DOX) accumulation or cytotoxicity. Upon incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA), HTB-123 cells retained less DOX than control cells and were sensitive to the efflux blockers verapamil or dipyridamole. These observations suggest that 12-O-tetradecanoylphorbol-13-acetate-induced P-gp phosphorylation may be associated with induction of P-gp-mediated drug efflux in the HTB-123 cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Inhibition of protein tyrosine phosphatases PTP1B and CD45 by sulfotyrosyl peptides.

Sulfotyrosyl peptides corresponding to the known high-affinity substrate phosphotyrosyl peptide sequences in casein and the autophosphorylation sites of insulin receptor and EGF receptor were investigated as inhibitors of protein tyrosine phosphatases PTP1B and CD45. These peptides inhibit both PTP1B and CD45 in the micromolar range competitively and reversibly. The elements required for inhibition were investigated by truncation and substitution of these peptides. Acidic residues N-terminal to the sulfotyrosyl residues are essential for high-affinity binding to PTP1B. The recognition elements required for inhibition of PTP1B and CD45 are different and this suggests the possibility of identifying selective active-site-directed inhibitors for these enzymes.

Binding Sites↗

PubMed1998

Affinity selection from peptide libraries to determine substrate specificity of protein tyrosine phosphatases.

Affinity selection from peptide libraries is a powerful tool that has been used for determining the sequence specificities of a number of enzymes and protein binding domains, including protein kinases, src homology 2 domains, and PDZ domains. We have extended this approach to protein tyrosine phosphatases using peptide libraries containing a nonhydrolyzable phosphotyrosine analog, difluorophosphonomethylphenylalanine. A size-exclusion method is used to separate enzyme-peptide complexes from free peptide, providing several advantages over the traditional immobilized protein affinity column approach. In addition, the feasibility of using mass spectrometric detection to quantitate peptides rapidly and reproducibly is demonstrated as an alternative to quantitation by peptide sequencing. The validity of this analysis is demonstrated by synthesizing individual peptides and comparing their affinity for enzyme with the predictions from the affinity selection process. As a model for these studies the protein tyrosine phosphatase PTP1B is used, providing additional insights into the sequence specificity of this enzyme. In particular, a selection for aromatic amino acids at the pY - 1 position (immediately N-terminal to the phosphotyrosine), as well as a broad pY + 1 selectivity, is observed in addition to the general preference for acidic residues N-terminal to the phosphotyrosine. The approach described here should prove applicable to protein tyrosine phosphatases in general as well as for the study of nonpeptidyl combinatorial libraries.

Amino Acid Sequence↗

PubMed1998

The specificity of the N-terminal SH2 domain of SHP-2 is modified by a single point mutation.

SH2 domains are small protein domains of approximately 100 amino acids that bind to phosphotyrosine (pY) in the context of a specific sequence surrounding the target pY. In general, the residues C-terminal to the pY of the binding target are considered most important for defining the binding specificity, and in particular the pY + 1 and pY + 3 residues (i.e., the first and third amino acids C-terminal to the pY). However, our previous studies with the SH2 domains of the protein tyrosine phosphatase SHP-2 [Huyer, G., Li, Z. M., Adam, M., Huckle, W. R., and Ramachandran, C. (1995) Biochemistry 34, 1040-1049] indicated important interactions with the pY - 2 residue as well. In the SH2 domains of SHP-2, the highly conserved alphaA2 Arg is replaced by Gly. A comparison of the published crystal structures of the Src SH2 domain and the N-terminal SH2 domain of SHP-2 complexed with high-affinity peptides suggested that the alphaA2 Gly of SHP-2 creates a gap which is filled by the side chain of the pY - 2 residue of the bound peptide. It was predicted that replacing this Gly with Arg would alter or eliminate the involvement of the pY - 2 residue in binding. The alphaA2 Gly --> Arg mutant was constructed, and indeed, this mutant no longer required residues N-terminal to the target pY for high-affinity binding, making its specificity more like that of other SH2 domains. The alphaA2 Gly is clearly involved in directing the unusual requirement for the pY - 2 residue in the binding sequence of this SH2 domain, which has important implications for its in vivo targeting and specificity.

Amino Acid Sequence↗

PubMed1998

Naphthalenebis[alpha,alpha-difluoromethylenephosphonates] as potent inhibitors of protein tyrosine phosphatases.

A series of naphthalenebis(difluoromethylenephosphonates) were prepared and compared to their monosubstituted counterparts as inhibitors of the protein phosphatases, PTP1B, CD45 and PP2A. In general, the bissubstituted compounds were better inhibitors than the mono derivatives and some of these are among the most potent, nonpeptidyl inhibitors of protein tyrosine phosphatases, PTP1B and CD45, reported to date.

Catalytic Domain↗

PubMed1998

Potent non-peptidyl inhibitors of protein tyrosine phosphatase 1B.

The development of inhibitors of protein tyrosine phosphatases (PTPs) has recently been the subject of intensive investigation due to their potential as chemotherapeutics and as tools for studying signal transduction pathways. Here we report the evaluation of a variety of small molecule, non-peptidyl inhibitors of protein tyrosine phosphatase 1B (PTP1B), bearing the alpha, alpha-difluoromethylenephosphonic acid (DFMP) group, a non-hydrolyzable phosphate mimetic. A series of phenyl derivatives bearing a single DFMP group were initially surveyed. In general, these were not significantly more potent inhibitors than the parent compound, alpha, alpha-difluorobenzylphosphonic acid, with the exception being the meta-phenyl substituted species which decreased the IC50 by approximately 17-fold relative to alpha, alpha-difluorobenzylphosphonic acid. However, certain compounds bearing two DFMP moieties were very potent inhibitors. Some of these are among the most potent small molecule inhibitors of any PTP reported to date with the best one exhibiting a Ki of 1.5 microM. The structural basis for these results are discussed. One of the bis-DFMP inhibitors was examined in detail and it was found that the fluorines were essential for potent inhibition. Inhibition was independent of pH between pH 5.5-7.2 suggesting that both the mono and dianionic forms of the individual DFMP groups bind equally well. The trends observed in the inhibitory potency of these compounds with PTP1B were very similar to the trends observed by other workers on the K(m)'s of the analogous phenylphosphate substrates with rat PTP1. This indicates that studies of non-peptidyl substrates with rat PTP1 can be used as a guide for the development of human PTP1B inhibitors.

Enzyme Inhibitors↗

PubMed1997

Mechanism of inhibition of protein-tyrosine phosphatases by disodium aurothiomalate.

Disodium aurothiomalate (AuTM) has been used successfully in the treatment of various autoimmune and inflammatory disorders; however, the molecular target(s) for this agent remains unknown. The aim of this study was to investigate whether the activity of CD45, a protein-tyrosine phosphatase (PTP, EC 3.1.3.48) essential for antigen-receptor-mediated lymphocyte signaling, was modified by AuTM exposure. The effects of AuTM on the activities of CD45 and other PTPs were monitored in vitro by a continuous assay using the substrate fluorescein diphosphate. In addition, the inhibition of PTP1B by AuTM was determined using a novel binding assay that employed an optical biosensor (BIAcore). The experimental results are summarized here: AuTM inhibited CD45 activity with an IC50 of 1.2 +/- 0.1 microM, and inhibition was competitive with substrate. The effect of AuTM, however, was not restricted to CD45, as the cytoplasmic PTP (PTP1B) was also inhibited, with an IC50 of 3.6 +/- 0.2 microM. AuTM also blocked the binding of GST-PTP1B to an immobilized active site inhibitor: a non-hydrolyzable difluorophosphonomethyl phenylalanine-containing biotinylated hexapeptide. AuTM-inhibited CD45 could be reactivated by the addition of excess dithiothreitol. These findings indicate that AuTM may interact with the essential active site cysteine residue involved in the catalytic mechanism of PTPs. Thus, it is possible that some of the cellular effects of gold result from the inhibition of these important cell signaling molecules.

Binding, Competitive↗

PubMed1997

How does alendronate inhibit protein-tyrosine phosphatases?

Alendronate (4-amino-1-hydroxybutylidene 1,1-bisphosphonate) is a drug used in the treatment of osteoporosis and other bone diseases. The inhibition of protein-tyrosine phosphatases (PTPs) by alendronate suggests that PTPs may be molecular targets. As a clear understanding of the inhibition mechanism is lacking, our aim was to analyze the mechanism to provide further insight into its therapeutic effect. We show here that the inhibition of PTPs by alendronate in the presence of calcium followed first-order kinetic behavior, and kinetic parameters for the process were determined. Evidence is presented that the inhibition by alendronate/calcium is active site-directed. However, this process was very sensitive to assay constituents such as EDTA and dithiothreitol. Furthermore, the inhibition of PTPs by alendronate/calcium was eliminated by the addition of catalase. These observations suggest that a combination of alendronate, metal ions, and hydrogen peroxide is responsible for the inhibition of PTPs. The individual effects of alendronate, calcium, or hydrogen peroxide on the inactivation of CD45 were determined. Electrospray ionization mass spectrometry demonstrated that the mass of PTP1B increased by 34 +/- 2 units after the enzyme was inactivated with alendronate/calcium, due to the oxidization of the catalytic cysteine to sulfinic acid (Cys-SO2H). The inhibited PTP1B could be partially reactivated by treatment with reducing agents such as hydroxylamine (NH2OH) and N,N'-dimethyl-N, N'-bis(mercaptoacetyl)hydrazine, indicating the presence of other oxidized forms such as sulfenic acid (Cys-SOH). This further confirms that the inhibition is the result of oxidation of the catalytic cysteine. The relevance of this oxidative inhibition mechanism in a biological system is discussed.

Alendronate↗

PubMed1997

Flow cytometric analysis of P-glycoprotein expression and drug efflux in human soft tissue and bone sarcomas.

Twenty-two fresh surgical specimens of human sarcomas (soft tissue and bone) from 20 patients were analyzed by flow cytometry for the expression of drug resistance-related P-glycoprotein (P-gp) and cellular daunorubicin (DNR) accumulation with or without the presence of DNR efflux blockers. Single-cell suspensions prepared from the tumor specimens were analyzed by dual-color flow cytometry after reaction with MRK-16 (anti-P-gp) and anti-CD45 (pan-leukocyte) antibodies. MRK-16 reactivity of tumor cells was evaluated after exclusion of CD45-positive cells by electronic gates. Parallel samples were incubated with DNR alone or in combination with DNR efflux blockers, verapamil (VPL), or dipyridamole (DPD) for determination of cellular DNR accumulation and the effect of the efflux blockers. Extensive heterogeneity was observed in both P-gp expression and DNR accumulation of the tumor specimens examined. Eight of the 22 tumor specimens had significant numbers of P-gp-positive cells. In three of the eight P-gp-positive tumors, cellular DNR accumulation was significantly increased by co-incubation with the efflux blockers VPL or DPD. These results indicate that both quantitative and functional analysis of P-gp expression may be essential in determining the cellular drug resistance phenotype of tumor cells and its correlation with therapeutic outcome.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate.

Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition. Our results show that vanadate is a competitive inhibitor for the protein-tyrosine phosphatase PTP1B, with a Ki of 0.38+/-0.02 microM. EDTA, which is known to chelate vanadate, causes an immediate and complete reversal of the inhibition due to vanadate when added to an enzyme assay. Pervanadate, by contrast, inhibits by irreversibly oxidizing the catalytic cysteine of PTP1B, as determined by mass spectrometry. Reducing agents such as dithiothreitol that are used in PTP assays to keep the catalytic cysteine reduced and active were found to convert pervanadate rapidly to vanadate. Under certain conditions, slow time-dependent inactivation by vanadate was observed; since catalase blocked this inactivation, it was ascribed to in situ generation of hydrogen peroxide and subsequent formation of pervanadate. Implications for the use of these compounds as inhibitors and rationalization for some of their in vivo effects are considered.

Animals↗

PubMed1997

Perifollicular transgenic expression of human interleukin-1 receptor antagonist protein following topical application of novel liposome-plasmid DNA formulations in vivo.

Expression plasmid DNA for the human interleukin-1 receptor antagonist (IL-1ra) protein was formulated with nonionic:cationic (NC) liposomes or phosphatidylcholine:cationic (PC) liposomes and applied to the auricular skin of hamsters in single- and multiple-dose protocols. Confocal microscopy identified delivery of plasmid DNA proximal to perifollicular cells, and successful transfection of perifollicular cells was identified by immunohistochemistry and ELISA. Skin treated for 3 days with the NC liposomes had statistically significant levels of transgenic IL-1ra present for 5 days post-treatment. Expression of transgenic IL-1ra was specific to areas of skin treated with NC liposomes but not PC liposomes. The results indicate that the NC liposomes can deliver expression plasmid DNA to perifollicular cells and mediate transient transfection in vivo.

Administration, Topical↗

PubMed1996

Rapid adaptive camouflage in tropical flounders.

Despite the commonly held view that flatfish can change their surface markings to match their background pattern, there have been few systematic studies and it has recently been claimed that their capacity for such adaptive changes is minimal. Here we show that the tropical flatfish Bothus ocellatus can achieve pattern-matching with surprising fidelity. By adjusting the contrast of different sets of 'splotches' of different grain size (or spatial frequency) on the skin, the fish can blend into a wide range of background textures in just 2-8 seconds.

Adaptation, Physiological↗

PubMed1996

Topical delivery of erythromycin from various formulations: an in vivo hairless mouse study.

Topical products containing erythromycin, a macrolide antibiotic with poor aqueous solubility, are usually formulated as high alcohol content solutions or gels. In this study, we evaluated the deposition of erythromycin base into various strata of hairless mouse skin following topical in vivo application from various low- and nonalcoholic formulations. The formulations tested included nonionic liposomal formulation composed of glyceryl dilaurate, cholesterol, and polyoxyethylene-10-stearyl either at a weight ratio of 57:15:28, two nonionic oil-in-water (o/w) liposomal emulsions containing isopropyl myristate or light mineral oil as the oil phase, a conventional o/w emulsion, a 40% hydroalcoholic solution, and two commercially available topical products. Eight hours after topical administration of these formulations, the efficiency of uptake of erythromycin into the living skin strata was in the order: liposomal isopropyl myristate emulsion > > liposomal mineral oil emulsion > > nonionic liposomes approximately Emgel approximately Theramycin-Z > > conventional emulsion > > hydroalcoholic solution. Alcohol-free liposomal systems are shown to be as efficient as high alcohol content products in facilitating permeation of erythromycin through the stratum corneum into living skin tissue.

Administration, Cutaneous↗

PubMed1995

Expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes during cell cycle traverse.

The expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes was analyzed in cell cycle phase enriched populations of doxorubicin-resistant murine leukemic P388/R-84 cells. Flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation and staining with anti-BrdU antibodies was used to confirm the purity of cell cycle phase enriched populations obtained by centrifugal elutriation. Doxorubicin (DOX) and daunorubicin (DNR) accumulation was significantly lower in S-phase cells, and coincubation with verapamil (VPL) or chlorpromazine (CPZ) enhanced DOX and DNR accumulation more in S-phase than in G1- and G2/M-phase cells. While the cellular content of mdr-1 and topoisomerase II mRNAs changed, GST pi mRNA content remained constant during the cell cycle. S-phase cells had about 3-fold higher mdr-1 mRNA content than G1- and G2/M-phase cells. In G1 cells, P-glycoprotein expression, as determined by C219 monoclonal antibody, was 12% less than that of S and G2/M cells. Topoisomerase II mRNA content increased with the progression of cell cycle and peaked in G2/M cells. These observations suggest that cell cycle stage related changes in expression of drug resistance markers may have a major bearing on chemosensitivity of drug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

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↗