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Biomedical subjects

R Gopalakrishna

Publications and source records attributed to R Gopalakrishna.

At least 55 records · Page 3Linked to original sources
PubMed1987

Isolation of a myofibroblast growth factor from human breast carcinoma cell lines.

Conditioned media of a series of well-established human breast carcinoma cell lines were screened for mitogenic activity on human myofibroblasts. Whereas all carcinoma lines derived from desmoplastic ductal breast carcinoma primaries exhibited moderate to high levels of mitogenic activity, the single line derived from a non-desmoplastic (medullary) carcinoma exhibited low activity. Levels of mitogenic activity were independent of estrogen receptor status and estrogen/antiestrogen treatment. Fractionation of the conditioned media revealed a cationic, hydrophobic mitogenic factor of M.W. 25,000. The factor did not stimulate the growth of endothelial or carcinoma cells nor the growth of NRK fibroblasts in soft agar.

Animals↗
PubMed1987

Susceptibility of protein kinase C to oxidative inactivation: loss of both phosphotransferase activity and phorbol diester binding.

Exposure of protein kinase C to low concentrations of either N-chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF-7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane-associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation.

Animals↗
PubMed1987

Increased invasion and spontaneous metastasis of BL6 melanoma with inhibition of the desmoplastic response in C57 BL/6 mice.

BL6 melanoma cells injected s.c. in 18-month C57 BL/6 mice elicit a markedly fibrotic response similar in myofibroblast and collagen composition to that characterizing the desmoplastic response of human breast carcinoma. This host response can be quantitated by measuring hydroxyproline (total collagen) and incorporation of i.p.-injected [14C]proline into collagenase-sensitive protein (new collagen synthesis). Inhibition (70%) of the desmoplastic response can be achieved by daily injections of L-3,4-dehydroproline. Inhibiting the response in this manner promotes local invasion of tumor and increases the incidence of spontaneous pulmonary metastasis. 10(5) BL6 melanoma cells produce tumor nodules with a mean diameter of 1.5 +/- 0.5 cm and mean collagen content of 36 +/- 15 mg/g wet tissue at 4 weeks and 10% incidence of pulmonary metastasis at 7 weeks. L-3,4-dehydroproline produces nodules with a mean diameter of 2.3 +/- 0.5 cm and mean collagen content of 12 +/- 2 mg/g with a 40% incidence of metastasis. L-3,4-dehydroproline exerts a selective effect on myofibroblast collagen synthesis in vitro and no effect on [3H]thymidine uptake, doubling time, and viability of BL6 cells and myofibroblasts. Furthermore, this drug exerts no effect on BL6 invasion and metastasis in 6-week C57 BL/6 mice, hosts which exhibit a negligible desmoplastic response.

Animals↗
PubMed1987

An experimental model for studying the desmoplastic response to tumor invasion.

BL6 melanoma cells injected subcutaneously in 18-month-old syngeneic C57BL/6 mice elicit a marked fibrotic response highly similar in myofibroblast composition and Type V collagen content to that characterizing the desmoplastic response of human carcinomas. This host response can be quantitated in vivo by measuring both hydroxyproline content (total collagen) and incorporation of intraperitoneally injected [14C] proline into collagenase-sensitive protein (new collagen synthesis). 70% inhibition of the response can be achieved with daily L-3,4-dehydroproline. The response can be similarly quantitated in vitro in explants of desmoplastic tumor tissue. The model allows for subsequent investigations of the effects of the desmoplastic response on tumor invasion and metastasis.

Animals↗
PubMed1986

Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C.

One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.

Animals↗
PubMed1986

Hydrophobic association of calpains with subcellular organelles. Compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues.

Calpains I and II isolated from diverse tissues possess both Ca2+-independent, and Ca2+-dependent accessible hydrophobic regions. Possible subcellular organelle association of calpains involving these hydrophobic regions was studied. By homogenizing rat tissues directly in Ca2+ (50 microM), about 30-60% of the cytosolic calpain I and II activity reversibly associated with isolated subcellular fractions (microsomal greater than plasma membrane greater than nuclear). After binding to the particulate fraction, calpain II converted to a calpain I-like form exhibiting stronger Ca2+-independent binding to phenyl-Sepharose and a lower Ca2+ requirement for optimal activity. However, it retained its DEAE-cellulose chromatographic pattern, and precipitated with monospecific anti-calpain II antibodies. Although purified calpastatin (endogenous inhibitor) is known to form a Ca2+-dependent complex with calpains, it was not able to reverse the binding of calpains to the particulate fraction upon short incubation. It was, however, effective in blocking calpain binding when the isolated cytosolic fraction or a mixture of purified calpain and calpastatin was preincubated in the presence of Ca2+, and then added to the particulate fraction. Extraction of tissues under controlled conditions revealed that in fact calpains are already loosely associated with subcellular organelles even in the absence of Ca2+. This is the reason why in the crude homogenates with the addition of Ca2+, calpains strongly bind to the particulate fraction without interference by cytosolic calpastatin. Although calpastatin by complexing initially to calpain can prevent the association of this protease with subcellular organelles, it cannot dissociate calpains already bound to these subcellular fractions. By prior Ca2+-independent association with the hydrophobic proteins present in the subcellular fractions, calpains overcome the 3- to 30-fold inhibitory excess of calpastatin in tissues.

Animals↗
PubMed1985

Quantitation of tissue calpain activity after isolation by hydrophobic chromatography.

A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl-Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes.

Animals↗
PubMed1985

Rapid purification of calcium-activated protease by calcium-dependent hydrophobic-interaction chromatography.

Both low Ca2+- and high Ca2+-requiring forms of Ca2+-activated protease (calpains I and II) were found to bind to phenyl-Sepharose in a calcium-dependent manner, suggesting that both enzymes expose a hydrophobic surface region in the presence of Ca2+. Inclusion of leupeptin in column buffers prevented the loss of activity during hydrophobic-interaction and substrate-affinity chromatography. Under these conditions calpain II (high calcium-requiring form) was rapidly purified from bovine brain and rabbit skeletal muscle using successive phenyl-Sepharose and casein-Sepharose columns.

Animals↗
PubMed1985

Isolation of S-100 binding proteins from brain by affinity chromatography.

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.

Animals↗
PubMed1985

The effects of chemical modification of calmodulin on Ca2+-induced exposure of a hydrophobic region. Separation of active and inactive forms of calmodulin.

Native calmodulin binds four calcium ions per molecule and exhibits strong Ca2+-dependent binding to phenyl-Sepharose. In contrast, calmodulin inactivated by oxidation of methionine residues or by deamidation binds fewer calcium ions (two per molecule) and shows relatively weak interaction with phenyl-Sepharose. Calmodulin inactivated by modification of lysine residues still is able to bind four calcium ions per molecule and shows strong binding to phenyl-Sepharose similar to native calmodulin. The results suggest that complete exposure of calmodulin's hydrophobic region occurs only after the binding of four ions of calcium to the calmodulin molecule. Thus, phenyl-Sepharose hydrophobic interaction chromatography might be used to separate active calmodulin from inactive forms of calmodulin obtained by oxidation or heat treatment for prolonged periods. As an example, phenyl-Sepharose chromatography can be used to separate free iodide and inactivated species of calmodulin readily from the active, iodinated form of calmodulin following iodination.

Animals↗
PubMed1983

Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins.

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗