Biomedical subjects

S R Kain

Publications and source records attributed to S R Kain.

44 records · Page 3Linked to original sources

PubMed1993

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate stimulates the dephosphorylation of mitochondrial ferredoxin in cultured chick kidney cells.

The tumor promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), presumably through activation of protein kinase C, decreases the production of 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3] and increases that of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] by primary cultures of chick kidney cells. We have previously shown that the regulation of the cellular output of 1,25(OH)2D3 and 24,25(OH)2D3 by PTH and 1,25(OH)2D3 can be quantitatively accounted for by altered hydroxylase activities within isolated mitochondria. In the present paper, we examined the effects of TPA and 1-oleoyl-2-acetyl-glycerol (OAG) on the state of mitochondrial protein phosphorylation and on 25-hydroxyvitamin D3 [25(OH)D3] metabolism. There was a good correlation between 25(OH)D3- 1 alpha- and 24-hydroxylase activities in mitochondria isolated from cells pretreated with either TPA or OAG and the pattern of 1- and 24-hydroxylation of 25(OH)D3. The most notable change in protein phosphorylation in the molecular mass range of 10-60 kilodaltons (kDa) was a dramatic decrease in the phosphorylation of a 12.5-kDa mitochondrial matrix protein after treatment of kidney cells with TPA or OAG. The amino acid composition of the 12.5-kDa protein was similar to bovine and human ferredoxins and it comigrated with bovine and human ferredoxins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 12.5-kDa phosphoprotein was immunoprecipitated specifically by an antipeptide polyclonal antibody for chick ferredoxin. The dephosphorylation of ferredoxin in response to TPA was both rapid and transient, with the phosphate content of the 12.5-kDa protein reduced by 70% after a 5-min exposure and returning to control levels by 20 min. A similar transience was observed with regard to the rapid effects of TPA on 1 alpha-hydroxylase activity, again showing maximal inhibition at 5 min. The results of our studies are consistent with the idea that ferredoxin phosphorylation plays a role in the regulation of steroid hydroxylation.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1992

Disruptions in intracellular membrane trafficking and structure preclude the glucocorticoid-dependent maturation of mouse mammary tumor virus proteins in rat hepatoma cells.

We have previously shown that glucocorticoids regulate the trafficking and processing of mouse mammary tumor virus (MMTV) proteins in viral-infected M1.54 rat hepatoma cells. To examine the role of intracellular membrane integrity on MMTV protein maturation, brefeldin A (BFA) was utilized to disrupt membrane flow between the endoplasmic reticulum and Golgi. Immunoprecipitation and immunofluorescence microscopy revealed that in the presence of dexamethasone, BFA inhibited the proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins. Glycosidase digestion and inhibitors of protein glycosylation confirmed that the observed differences in apparent sizes of MMTV glycoprotein products are due to BFA-induced changes in oligosaccharide processing. BFA treatment inhibited the proteolytic processing of the MMTV phosphoprotein precursor, which normally associates with the cytoplasmic face of intracellular membranes. Similarities in salt extraction efficiency revealed that BFA did not affect the membrane affinity of the uncleaved phosphorylated precursor. In a complementary approach, proteolytic processing of the phosphorylated polyprotein did not occur in glucocorticoid-treated HTC cells transfected with a mutant MMTV provirus encoding a normal phosphorylated precursor, but which express a truncated MMTV glycoprotein missing its transmembrane domain and cytoplasmic tail. These results suggest that the MMTV glycoproteins and phosphoproteins may interact at a late step in the transport pathway in a manner required for their mutual processing in response to glucocorticoids and establishes the importance of functional interactions with intracellular membranes for maturation of the cytoplasmic MMTV phosphoproteins.

Animals↗

PubMed1992

Differential transport and processing of variant mouse mammary tumor virus glycoproteins.

The transport and proteolytic processing of two individual gene isolates of the mouse mammary tumor virus (MMTV) glycoprotein were compared in transfected rat HTC hepatoma cells. Plasmids were constructed such that the MMTV glycoprotein genes were constitutively expressed from the promoter within the Rous Sarcoma Virus 5' Long Terminal Repeat in the absence of other MMTV proteins. An isolate of the GR strain MMTV glycoprotein was efficiently transported and processed resulting in the localization of MMTV glycoproteins at the cell surface and in the extracellular environment. Moreover, the kinetics of acquisition of endoglycosidase H resistant oligosaccharide side chains and the rate of endoproteolytic cleavage of the glycosylated polyprotein expressed in transfected cells were virtually identical to that observed in viral-infected rat hepatoma cells. In contrast, a natural variant of the C3H strain MMTV glycoprotein expressed in transfected cells was retained in an intracellular compartment by a heavy chain binding protein (BiP)-independent pathway in an endoglycosidase H sensitive and uncleaved form. This MMTV glycoprotein isolate was retained early in the exocytic pathway and displayed a half-life of approximately 45 min in transfected cells. Only a minor fraction of the expressed C3H variant glycoprotein was detected at the cell surface but was not externalized. Our results suggest that the variant C3H MMTV glycoprotein contains one or more mutations that preclude its efficient transport through the exocytic pathway.

Animals↗

PubMed1991

Altered effects of glucocorticoids on the trafficking and processing of mouse mammary tumor virus glycoproteins constitutively expressed in rat hepatoma cells in the absence of nonglycosylated viral components.

We have documented previously that glucocorticoid hormones modulate the posttranslational localization of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the viral-infected M1.54 rat HTC hepatoma cell line. To determine whether glucocorticoids affect the trafficking of individually synthesized MMTV glycoproteins, HTC cells were transfected with a constitutively expressed MMTV glycoprotein gene lacking the viral phosphoprotein and polymerase genes. This construct also allows equivalent levels of MMTV glycoproteins to be compared in the presence or absence of glucocorticoids. Indirect immunofluorescence and immunoprecipitation of radiolabeled cells revealed that in transfected cells the transmembrane MMTV glycoproteins are efficiently expressed, transported to the cell surface, and proteolytically cleaved in the presence or in the absence of the synthetic glucocorticoid dexamethasone. Cell surface immunoprecipitation of [35S]methionine-labeled cells showed that the level of plasma membrane gp78 appeared to be stimulated 2-fold after dexamethasone treatment, even though fluorescence-activated cell sorting revealed no discernible change in the total concentration of cell surface MMTV glycoproteins. Analysis of oligosaccharide side chain maturation through a pulse-chase radiolabeling revealed that the rate of rough endoplasmic reticulum-Golgi transport was essentially identical in dexamethasone-treated and untreated transfected cells and was similar to that observed in dexamethasone-treated M1.54 cells. Thus, in contrast to viral-infected hepatoma cells, mostly constitutive cellular machinery mediates the trafficking and maturation of cell surface MMTV glycoproteins expressed outside of the proviral context. Taken together, our results suggest that the glucocorticoid-stimulated synthesis of nonglycosylated viral components may contribute to or be responsible for the regulated trafficking of MMTV glycoproteins observed in viral-infected rat hepatoma cells.

Amino Acid Sequence↗

PubMed1990

Quantitation of proteins bound to polyvinylidene difluoride membranes by elution of coomassie brilliant blue R-250.

A rapid and simple method for the quantitation of stained proteins bound to polyvinylidene difluoride (PVDF) membranes via the elution of Coomassie brilliant blue R-250 is described. A mixture of standard proteins was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Spectrophotometric analysis of dye eluted from protein bands in the range of 0.5-10 micrograms gave a linear change in the absorbance at 595 nm. Maximal absorbance readings were attained following 5 min of dye elution, and the readings remained unchanged for elution times up to 60 min. The method requires no unusual reagents or equipment, is suitable for the analysis of multiple samples, and does not consume the protein in the process of quantitation. This technique provides a useful means for the quantitation of proteins bound to PVDF membranes prior to amino acid sequence determination, immunological analysis, or other biochemical characterizations.

1-Propanol↗

PubMed1989

25-Hydroxyvitamin D3 metabolism in mitochondria from primary renal cultures.

Previous in vitro studies concerning the renal metabolism of 25-hydroxyvitamin D3 (25(OH)D3) to form 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25R-dihydroxyvitamin D3 (24,25(OH)2D3) have utilized intact cell systems. In reflecting upon the possible mechanisms by which hormonally induced changes in the production of 1,25(OH)2D3 and 24,25(OH)2D3 may be brought about, we asked whether altered mitochondrial hydroxylase activities can quantitatively account for changes in the total cellular output of these steroids. Our objective was to delineate between extramitochondrial processes (e.g. altered substrate delivery), and those events restricted to the renal mitochondria (altered hydroxylase activities). We have examined the effect of pretreating primary cultures of chick kidney cells with either 1,25(OH)2D3 or parathyroid hormone (PTH) on 25(OH)D3-hydroxylase activities present in subsequently isolated mitochondria. Pretreatment with 10(-7) M 1,25(OH)2D3 reduced 1 alpha-hydroxylase activity in both cells and mitochondria to approximately 60% of control values by 1 h, and to 25-30% by 2 h. The effect of PTH (10 ng/ml) in both mitochondrial and whole cell preparations was an approximate 40% increase in measured 1 alpha-hydroxylase activity. 10 microM forskolin (FSK) elicited an approximate 2-fold increase in 1,25(OH)2D3 production. Reciprocal effects were observed with respect to 24-hydroxylase activity in both whole cell and mitochondrial preparations in response to exogenous 1,25(OH)2D3, PTH, and FSK. The findings demonstrate that these hormones initiate intracellular events which lead directly to altered 25(OH)D3 1 alpha- and 24-hydroxylase activities within the renal mitochondria.

Animals↗

PubMed1988

Effect of vitamin D status on chick kidney proteins: detection of a 45-kilodalton mitochondrial protein suppressed by vitamin D.

Two-dimensional polyacrylamide gel electrophoresis along with L-[35S]methionine radiolabeling studies were used to examine the effect of chronic vitamin D status on the composition and relative abundance of chick kidney proteins. Comparison of silver-stained gels revealed no extensive differences in either the electrophoretic mobility or the amounts of kidney proteins present in the mitochondrial fraction from vitamin D-replete and vitamin D-deficient chicks. A similar result was obtained in studies with L-[35S]methionine-labeled proteins. Vitamin D deficiency specifically elevated levels of a 45-kilodalton mitochondrial protein (pI 5.0 to 5.5) by approximately 5- to 12-fold relative to amounts present in vitamin D-replete tissue. This protein could not be detected in postmitochondrial supernatant fractions and was only faintly visible in crude kidney homogenates. The specificity of the observed suppression of this 45-kilodalton protein by vitamin D suggests that it may play an important role in renal functions influenced by the vitamin D endocrine system.

Animals↗

PubMed1984

Selenium modified mutagenicity and metabolism of benzo[a]pyrene in an S9-dependent system.

Selenium added to the incubation mix containing rat-liver S9 modified both the metabolism and mutagenicity of benzo[a]pyrene (BaP) and several of its metabolites. Selenium (Na2SeO3) inhibited the S9-dependent mutagenic effects of BaP on Salmonella typhimurium strain TA100 as indicated by the number of histidine-dependent revertants counted. This inhibition was concentration-dependent over a range of 12.5 to 100 ppm. When used as the substrate the BaP metabolites 7,8-dihydrodiol, 9,10-dihydrodiol and 3-hydroxy also produced significantly fewer revertants in TA100 when selenium was included in the incubation mix. High-performance liquid chromatographic analysis of metabolites from S9-dependent metabolism of BaP indicated that selenium inhibited the formation of 3-hydroxy-BaP, 9,10-dihydrodiol, 7,8-dihydrodiol, 1,3- and 3,6-quinone. Eluting samples on an alumina column to isolate the conjugated metabolites showed that selenium caused 12% less binding to glucuronides, no significant differences in binding to sulfate esters or glutathione but the amount of unmetabolized BaP and unconjugated metabolites was increased by 48%. These results suggest that selenium inhibits S9-dependent BaP metabolism therefore reducing the mutagenic effects of this compound.

Animals↗