Search PubMed⌕ Search

Biomedical subjects

A Subramanian

Publications and source records attributed to A Subramanian.

At least 55 records · Page 3Linked to original sources
PubMed1996

Rate limitations in posttranslational processing by the mammary gland of transgenic animals.

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

Amino Acid Sequence↗
PubMed1995

The MAP kinase signal transduction pathway is activated by the endogenous cannabinoid anandamide.

Anandamide is an endogenous ligand for delta 9-tetrahydrocannabinol (THC) receptors. Incubation of cultured cells with anandamide or THC causes increased arachidonic acid release and eicosanoid biosynthesis. Here we demonstrate that the MAP kinase signal transduction pathway contributes to this response. Treatment of WI-38 fibroblasts with anandamide causes increased MAP kinase activity and increased phosphorylation of the arachidonate-specific cytoplasmic phospholipase A2 (cPLA2). Significantly, MAP kinase phosphorylates and activates cPLA2 [Lin, et al., Cell, 72 (1993) 269-278]. The MAP kinase signal transduction pathway may therefore mediate the effects of anadamide on cPLA2 activation and arachidonic acid release.

Arachidonic Acid↗
PubMed1995

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures.

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.

Animals↗
PubMed1995

Cannabinoid photolabelling of a G-protein gamma-subunit in mouse peritoneal cells.

Binding and photoactivation of a cannabinoid-derived ligand to intact mouse peritoneal cells has resulted in the labelling of a G-protein gamma-subunit. The assignment of structure is based on the product's physical characteristics and its ability to react with a polyclonal antiserum raised against the partial amino acid sequence for a gamma-subunit expressed in spleen cells. The binding characteristics of the ligand to the cells suggests that this gamma-subunit, and its associated alpha and beta subunits, are located in close proximity to one of the transmembrane cannabinoid receptors. Our findings further suggest a possible experimental approach to identifying receptor-G-protein complexes in intact cells.

Amino Acid Sequence↗
PubMed1995

A STAT factor mediates the sexually dimorphic regulation of hepatic cytochrome P450 3A10/lithocholic acid 6 beta-hydroxylase gene expression by growth hormone.

Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes.

Amino Acid Sequence↗
PubMed1995

MR urography: technique and application.

PURPOSE: To evaluate a magnetic resonance (MR) technique for depicting the kidneys and urinary tract. MATERIALS AND METHODS: Fourteen patients with urinary tract obstruction and 20 without obstructions were examined with a modified, heavily T2-weighted fast spin-echo pulse sequence (MR urography). In addition, six healthy volunteers underwent modified MR urography with intravenous administration of furosemide and ureteral compression prior to imaging. RESULTS: MR urography provided high-resolution images of the kidneys and urinary tract in all patients with obstruction with intact collecting systems. Anatomic anomalies were depicted in two patients. Intraluminal neoplasia was well demonstrated in both obstructed and nonobstructed systems (n = 4). Furosemide-enhanced MR urography provided fine anatomic and functional detail of both the kidneys and urinary tract (n = 6). CONCLUSION: MR urography may provide an alternative to more conventional urinary tract imaging techniques. It does not require ionizing radiation or iodinated contrast material. When combined with furosemide and ureteral compression, MR urography provides fine detail and reflects function.

Adult↗
PubMed1994

Phospholipase participation in cannabinoid-induced release of free arachidonic acid.

The exposure of cells in culture to cannabinoids results in a rapid and significant mobilization of phospholipid bound arachidonic acid. In vivo, this effect has been observed as a rise in eicosanoid tissue levels that may account for some of the pharmacological actions of delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid. Fluoroaluminate pretreatment of mouse peritoneal cells potently reduced the cannabinoid response, while promoting arachidonate release on its own, consistent with earlier observations that this effect may be a receptor/G-protein-mediated process. Further support for receptor mediation was the demonstration of saturable, high-affinity cannabinoid binding in these cells. THC potency was reduced in the presence of ethanol, and was accompanied by significant increases in phosphatidylethanol (PdEt) levels, a unique product of phospholipase D (PLD) activity. THC-dependent arachidonate release was reduced partially in similar amounts by either propranolol or wortmannin, further implicating PLD as a mediator of THC action. A central role for diacylglyceride (DAG), a secondary product of PLD metabolism, in this THC-induced process, both as a source of arachidonate and as a stimulator of protein kinase C (PKC), is supported by the data in this report. Cells exposed to phorbol ester for 18 hr prior to THC challenge became less responsive, indicating a possible role for PKC. The involvement of PKC further suggests participation by phospholipase A2 (PLA2) whose activity may be regulated by the former. Treatment of cells with interleukin-1 alpha, an agent known to elevate PLA2 levels, caused an increase in the THC response, supporting a role for this enzyme in the release reaction. Direct evidence, by immunoblotting, for the activation and phosphorylation of PLA2 by THC was also obtained. In summary, the evidence presented in this report indicates that THC-induced arachidonic acid release occurs through a series of events that are consistent with a receptor-mediated process involving the stimulation of one or more phospholipases.

Animals↗
PubMed1992

High-level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C.

Transgenic pigs were generated that produced human protein C in their milk at up to 1 g/liter. The gene construct was a fusion gene consisting of the cDNA for human protein C inserted into the first exon of the mouse whey acidic protein gene. These results demonstrate that the mouse whey acidic protein gene contains regulatory elements that can direct cDNA expression at high levels in the pig mammary gland. Recombinant human protein C that was produced at about 380 micrograms/ml per hr in transgenic pig milk possessed anticoagulant activity that was equivalent to that of protein C derived from human plasma. These studies provide evidence that gamma-carboxylation can occur at high levels in the mammary gland of a pig.

Animals↗
PubMed1992

A possible modulatory influence of melatonin on representative phase I and II drug metabolizing enzymes in 9,10-dimethyl-1,2-benzanthracene induced rat mammary tumorigenesis.

The oncosuppressive effect of melatonin on 9,10-dimethyl-1,2-benzanthracene (DMBA) induced rat mammary tumorigenesis led us to assess its possible modulatory influence on representative hepatic and mammary drug metabolizing enzymes in DMBA treated female Holtzman rats, reared in short and long photoperiods. Melatonin treated rats in either photoperiod showed a significant induction in hepatic and mammary levels of glutathione (GSH) and cytosolic activities of glutathione S-transferase (GST) when compared with the corresponding controls, along with a significant drop in hepatic microsomal contents of cytochromes b5 and P450. This induction of GSH and GST, and depletion of cytochromes b5 and P450 by melatonin may possibly be related to its anticarcinogenic potential in this tumor model.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Effect of neonatal pineal ablation on estradiol receptors in mammary glands of rats housed under varying photoperiods.

Estradiol receptors (ER) in mammary glands of female Holtzman rats, either intact or neonatally pinealectomised and housed in 10:14 or 24:0 L:D (light:dark) schedule were studied at 30, 40, 50 +/- 5 and 65 +/- 5 days of age. Whereas ER were detectable only at the age of 60-65 days in intact rats housed in 10:14 L:D, they were present as early as 30 days onwards in the pineal ablated group. In the 24:0 L:D pinealectomised group, though mammary gland ER were maximum around 40 days of age and temporarily undetectable around day 50, they had stabilized around the age of 60-65 days. The data demonstrates for the first time, the modulation of ER in rat mammary glands in response to varying photoperiods as well as pineal ablation. Earlier reports on incidence of chemically induced mammary tumours have been compared to the receptor modulation.

Animals↗
PubMed1991

Denatured autologous muscle graft in leprosy.

Autologous muscle grafts were used to repair 12 mixed peripheral nerves (9 posterior tibial, 3 median) in 10 patients with leprosy who had total anaesthesia and analgesia of the area supplied by the nerve. Postoperatively, 7 patients reported improved sensation in the foot or hand, with a return of vibration sense and joint position sense in 11 and of perception of a 10 g pin in 5; the ability to sweat in the affected area was also restored in 7.

Adolescent↗
PubMed1991

Suppressive effect by melatonin on different phases of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced rat mammary gland carcinogenesis.

This comprehensive study examines the influence of oral melatonin on the initiation and promotion phases of DMBA-induced mammary tumorigenesis in intact and pinealectomized female Holtzman rats reared in short (light:dark schedule L:D 10:14) and long (L:D 24:0) photoperiods. Melatonin administration in the initiation phase significantly suppressed tumor incidence only in intact animals reared in both photoperiods, indicating that the presence of the pineal was obligatory. On the other hand, during the promotion phase, irrespective of the presence or absence of the pineal, the tumor-suppressive effect of exogenous melatonin was pronounced.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Melatonin, a suppressor of spontaneous murine mammary tumors.

The effect of prolonged, oral melatonin treatment on spontaneous mammary tumor development in female C3H/Jax mice was studied. Melatonin was administered at a dose of 25 micrograms/mouse/day from 21 to 44 days of age and 50 micrograms/mouse/day from day 45 onwards. By the age of 12 months, 62.5% of control animals developed tumors as opposed to 23.1% in the melatonin treated group (P less than 0.02). In both control and treated mice, the thoracic pairs of mammary glands were obviously more susceptible to spontaneous mammary tumor development, as at least 50% of the total tumors developed in this region. Reduction in submaxillary and pituitary gland weights of treated animals was observed at necropsy (P less than 0.001). Decreased serum 17-beta-estradiol (E2) levels in melatonin treated mice (P less than 0.05) and a marked reduction in [3H]thymidine incorporation into DNA of melatonin-treated mammary glands (P less than 0.02) positively correlated with the sparse mammary gland development seen in these mice. These observations indicate that at the given dose level, melatonin modulates the degree of development of the mammary epithelium, subsequently reducing spontaneous mammary tumor incidence in these high risk mice.

Administration, Oral↗
PubMed1989

Preliminary investigation on antibacterial activity of some marine diatoms.

Seven species of marine diatoms were cultured under defined laboratory conditions. Air dried algal powder was used for extraction with different solvents in sequence. The algal extracts were tested against various bacteria by paper disc method. Nitzschia longissma, Skeletonema costatum and Biddulphia sinensis were effective against the test bacteria. Hemidiscus hardmannianus, Coscinodiscus centralis and Asterionella japonica showed moderate antibacterial activity. Pleurosigma elongatum exhibited no activity. The antibacterial substances were fully extracted by the organic solvents. Only in the active algal species, was activity noted in the aqueous extracts.

Anti-Bacterial Agents↗