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Biomedical subjects

S Seetharam

Publications and source records attributed to S Seetharam.

68 records · Page 4Linked to original sources
PubMed1986

An SV40-transformed xeroderma pigmentosum group D cell line: establishment, ultraviolet sensitivity, transfection efficiency and plasmid mutation induction.

Fibroblasts from a patient with xeroderma pigmentosum complementation group D were treated with Simian virus 40 to establish a transformed cell line suitable for studies of DNA-mediated gene transfer. After progressing through 2 crises, a stable line, XP6Be(SV40), was established and cultured for more than 1 year. This line retains the characteristic xeroderma pigmentosum ultraviolet hypersensitivity and is able to complement a SV40-transformed group A line when fused and assayed for ultraviolet radiation inhibition of colony-forming ability. XP6Be(SV40) expressed high levels of transfected chloramphenicol acetyltransferase activity (0.1 nmole X mg-1 X min-1) in a transient expression assay, showed stable expression of transfected gpt or neo genes (frequency 1-20 X 10(-5)), and permitted replication of the mutagenesis shuttle vector plasmid, pZ189. Ultraviolet treatment (500 J X m-2) of pZ189 prior to replication in XP6Be(SV40) resulted in a large reduction in plasmid yield (5% survival) and a 60-fold increase in the mutation frequency, reflecting the reduced ability of these cells to repair ultraviolet-damaged transfecting DNA. This cell line provides the opportunity to utilize transfection studies in cells with the xeroderma pigmentosum group D defect in excision repair.

Acetyltransferases↗
PubMed1985

Regulation of the biosynthesis of two distinct fatty acid-binding proteins in rat liver and intestine. Influences of sex difference and of clofibrate.

Two distinct fatty acid-binding proteins (FABPs) have been identified in rat intestine, gFABP (15,063 Da) which is confined to intestinal epithelium and hFABP (14,184 Da) which is found in both liver and intestine. We have examined the influence of sex difference and the effect of clofibrate, both of which affect cellular fatty acid metabolism and hFABP levels, on the concentration, and mRNA levels of both hepatic and intestinal FABPs. In the liver, hFABP concentration was approximately 2-fold greater in females and in clofibrate-treated males than in untreated male rats. These differences were not accompanied by changes in the fractional turnover of the polypeptide but rather by parallel increases in hFABP mRNA. In the intestine, the two FABPs exhibited different regulatory responses. Intestinal hFABP turnover was 33% greater in females than in males, whereas mRNA concentration was 50% greater. Thus, unlike hFABP in liver, there was no sex-related difference in the steady-state level of hFABP in intestine. However, clofibrate treatment, similar to its effects in the liver, doubled intestinal hFABP protein and mRNA concentration. In contrast to hFABP, neither gFABP protein nor mRNA concentration were sex dependent, whereas clofibrate produced only a modest increase in gFABP concentration without significantly changing gFABP mRNA levels. The results indicate that the influence of sex difference and the effect of clofibrate on hepatic fatty acid metabolism are both associated with changes in hFABP synthesis mediated pretranslationally. The differential response of hFABP and gFABP in intestine suggests that these proteins play distinct roles in the cellular metabolism of fatty acids.

Animals↗
PubMed1985

Protection against tumorigenesis by 3-methylcholanthrene in mice by beta-naphthoflavone as a function of inducibility of methylcholanthrene metabolism.

The noncarcinogenic enzyme inducer beta-naphthoflavone (beta-NF) causes an increase in both rate of activation and of detoxification of polycyclic aromatic hydrocarbon carcinogens in tissues of mice of induction-responsive strains. An experiment was carried out to test whether pretreatment with beta-NF would potentiate, protect against, or have no effect on the tumorigenicity of methylcholanthrene (MC) administered intragastrically to mice of varying responsiveness to induction of MC metabolism. The mouse strains used were C57BL/6, BALB/c, and C3H (inbred, highly responsive to induction), Swiss (random-bred, moderately responsive), and DBA/2 and AKR (inbred, nonresponsive). Twelve weekly treatments with 20 mg MC/kg intragastrically were preceded 24 h earlier each week by 150 mg beta-NF/kg i.p. or oil. Mice were killed when moribund or 1 year after start of treatment. During this period the beta-NF-pretreatment greatly reduced mortality due to cancer among the responsive inbred mice, by 100% for the C57BL/6, 89% for the BALB/c, and 65% for the C3H, compared with 50% for the Swiss, 23% for the DBA/2 and 0% for the AKR. There were significant reductions in MC-caused tumor incidences as a result of beta-NF pretreatment for: sarcomas, lymphomas, and forestomach and skin tumors for the C57BL/6 mice; sarcomas, mesotheliomas, and mammary carcinomas for the C3H mice; mesotheliomas for the BALB/c mice; sarcomas and tumors of the skin, forestomach, and lung for the Swiss mice; and lymphomas for the DBA/2 mice. beta-NF pretreatment did not cause an increase in the incidence of any type of tumor. These results are consistent with the conclusion that inducers of mixed function oxygenase activity generally provide protection against tumorigenesis by systemically administered polycyclic aromatic hydrocarbon carcinogens, probably by increasing rate of detoxification.

Age Factors↗
PubMed1984

Hepatic clearance of rat plasma intestinal alkaline phosphatase.

The mechanism and route of clearance of intestinal alkaline phosphatase from plasma have been studied in rats to define the magnitude of hepatic extraction and biliary excretion of the enzyme. Plasma clearance, tissue distribution, and biliary excretion of enzyme were followed after intravenous administration of physiological amounts of 125I-labeled rat intestinal alkaline phosphatase. The plasma disappearance curve was biphasic; the initial phase was rapid, during which 50% of injected enzyme was selectively extracted by the liver over 5 min. Less than 4% of total hepatic radioactivity was excreted into bile over 80 min; this was shown by chromatographic analysis to be degraded enzyme only. Rapid clearance of enzyme could be significantly slowed by injection of large amounts of mannan or N-acetylglucosamine-bovine serum albumin, but not by desialylated fetuin, demonstrating that clearance was probably mediated by mannose/N-acetylglucosamine-specific receptors. It is concluded that, under physiological conditions, rat plasma intestinal alkaline phosphatase is rapidly cleared from the circulation by the liver. However, biliary excretion of undergraded enzyme is negligible, and a physiologically significant enterohepatic circulation seems most unlikely.

Alkaline Phosphatase↗
PubMed1979

Pulmonary oat cell carcinomas. Expression of plasma membrane antigen correlated with presence of cytoplasmic neurosecretory granules.

A plasma membrane antigen highly associated with pulmonary oat cell carcinoma has recently been identified. Expression of this surface antigen was now examined in routine surgical pathology material from 32 primary lung tumors by immunostaining. The staining patterns of 17 tumors diagnosed as oat cell carcinoma by light microscopy were compared to those of 10 epidermoid carcinomas, four carcinoid tumors, and one lymphocytic lymphoma. In addition, when available, the oat cell carcinoma group was also studied by electron microscopy for demonstration of cytoplasmic neurosecretory granules. Twelve of the 17 oat cell carcinomas and one of the epidermoid carcinomas expressed the antigen. In this group of small cell tumors of the lung, aggrement was found between the expression of this immunochemical marker and the presence of neurosecretory granules in the cytoplasm of the tumors. Demonstration of this surface antigen in routine surgical pathologic material may be a useful aid in the diagnosis of these tumors. The failure of a group of tumors, which satisfied the light microscopic criteria for oat cell carcinoma to stain for the antigen, demonstrates heterogeneity within the oat cell carcinoma group.

Antigens, Neoplasm↗
PubMed1977

Identification of the Schwann cell as a peripheral nervous system cell possessing a differentiation antigen expressed by a human lung tumor.

Recent studies of the plasma membrane antigens of a human lung tumor (oat cell carcinoma) indicated that the tumor expressed at least two normal differentiation antigens undetectable in normal respiratory epithelium. One antigen was characteristic of certain endodermally derived epithelial cells of the digestive system; the other antigen was characteristic of certain neural crest-derived cells in the peripheral nervous system. The present studies were undertaken to identify the reactive cell type in the peripheral nervous system. Since similar cells in the rat peripheral nervous system expressed a cross-reactive form of this antigen, and since pure cultures of different rat nerve cell type were available, the following approach was possible. Cultures of pure neurons, pure Schwann cells, pure fibroblasts, neurons and Schwann cells, and neurons, Schwann cells, and fibroblasts were assayed for this antigen with rabbit anti-oat cell carcinoma plasma membrane antiserum absorbed with normal lung and liver. The indirect immunofluorescence method on both whole, viable cell and fixed cell substrates was used. Only Schwann cells expressed the antigen; Schwann cells in the presence of neurons expressed the antigen much more strongly than did pure Schwann cells. It was concluded that the oat cell carcinoma of the lung expressed a differentiation antigen present on Schwann cells.

Animals↗
PubMed1976

A plasma membrane antigen highly associated with oat-cell carcinoma of the lung and undetectable in normal adult tissue.

The plasma membrane antigens of an oat-cell carcinoma of the lung were studied to determine if any antigens absent from normal adult tissue could be identified. Rabbit and monkey antisera were prepared to a highly purified plasma membrane fraction of an oat-cell carcinoma of the lung. The specificities of the antisera were studied by the indirect immunofluorescence method on frozen section substrate. The rabbit antiserum, after absorption with normal lung, liver, colon and peripheral nerve homogenates and extracts, failed to react with any detectable normal adult tissue. The absorbed anti-serum did react with 7 of 7 oat-cell carcinomas of the lung, but failed to react with any of 7 adeno-carcinomas of the lung, 6 epidermoid carcinomas of the lung, 7 colon carcinomas, 8 breast carcinomas, 4 kidney carcinomas, and 1 pancreatic carcinoma. The unabsorbed monkey antiserum failed to react with any detectable normal adult tissue, and had a tumor reactivity pattern nearly identical to that of the absorbed rabbit antiserum. Thus similar results were obtained with antisera from two different species. It is concluded that oat-cell carcinomas of the lung express a plasma membrane antigen(s) undetectable in normal adult tissue and highly associated with this tumor type.

Adenocarcinoma↗
PubMed1976

Endodermally-derived and neural crest-derived differentiation antigens expressed by a human lung tumor.

The plasma membrane antigens of an undifferentiated small cell (oat cell) carcinoma of the lung were studied by the indirect immunofluorescence method on frozen section substrates with a rabbit antiserum prepared to the tumor plasma membrane fraction. After appropriate absorption of the antiserum, at least two differentiation antigens present on the tumor cells but undetectable on normal lung surface or glandular epithelial cells were identified. One antigen(s) was characteristic of certain normal, endodermally-derived epithelial cells of the digestive system, including those of colonic mucosa, hepatic ducts, pancreatic ducts and acini, and islets of Langerhans. The other antigen(s) was characteristic of certain normal, neural crest-derived cells in the peripheral nervous system, including cells in peripheral nerve, dorsal root ganglion, and anterior roots of the spinal cord; parasympathetic ganglion cells in the colon; and small nerves and nerve processes in the lung, colon, and skin. It was concluded that the presence of these differentiation antigens on the tumor cells resulted from the expression of gene products repressed in the normal cell of origin of the tumor.

Absorption↗
PubMedDate not supplied

The mechanism of elevated alkaline phosphatase activity after bile duct ligation in the rat.

Alkaline phosphatase activity in the liver and intestine increases after bile duct ligation, reportedly by increased enzyme synthesis. To ascertain the mechanism of this increased synthesis in the absence of a cDNA clone encoding the enzyme, we have estimated the concentration of liver and intestinal alkaline phosphatase mRNA by translational analysis. Monospecific antiserum to rat placental alkaline phosphatase was raised. The resulting antiserum precipitated two peptides of 53 and 56 kd after translation of liver poly(A) + RNA. The precipitation of both peptides was blocked by the single 64 kd placental alkaline phosphatase. Processing of the cell-free products by microsomal membranes produced peptides of 62 and 64 kd. Antiserum to rat intestinal alkaline phosphatase also identified two peptides as products of intestinal RNA translation. After bile duct ligation, we confirmed a transient 2-fold increase in alkaline phosphatase activity in the intestine and a more constant 7-fold increase in the liver. However, the alkaline phosphatase mRNA concentration remained unchanged in both organs. We conclude that increased alkaline phosphatase synthesis after bile duct ligation results from an enhanced rate of translation of mRNA.

Alkaline Phosphatase↗
PubMedDate not supplied

Single-chain immunotoxin fusions between anti-Tac and Pseudomonas exotoxin: relative importance of the two toxin disulfide bonds.

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light domains of the anti-IL2 receptor antibody, anti-Tac, are connected to each other by a peptide linker and then fused to PE40, a truncated form of Pseudomonas exotoxin (PE). This fusion protein has four disulfide bonds: one in each of the two variables domains, one in domain II (Cys 265-287), and one in domain Ib (Cys 372-379) of PE. To study the importance of the disulfide bonds of the toxin to the activity of single-chain immunotoxins, we constructed mutants in which either the cysteines in the toxin were changed to alanines or the amino acids 365-380 of PE were deleted. We began this study with anti-Tac(Fv)-PE40 and a more active variant, anti-Tac(Fv)-PE40KDEL, in which the carbonyl terminus is changed from REDLK to KDEL. From these proteins we made anti-Tac(Fv)-PE40(4)A and anti-Tac(Fv)-PE40KDEL4A, respectively, by converting cysteins at amino acids 265, 287, 372, and 379 of PE to alanines. This change resulted in a 20-100-fold loss of activity toward human target cells, but no significant change in binding affinity to p55. To determine the importance of the second toxin disulfide bond, we removed amino acids 365-380 from anti-Tac(Fv)-PE40, anti-Tac(Fv)-PE40KDEL, and anti-Tac(Fv)-PE40KDEL4A, resulting in anti-Tac(Fv)-PE38, anti-Tac(Fv)-PE38KDEL, and anti-Tac(Fv)-PE38KDEL2A, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP Ribose Transferases↗
PubMedDate not supplied

Antitumor interaction of short-course endostatin and ionizing radiation.

PURPOSE: The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR). Endostatin was injected to coincide with fractionated radiotherapy. METHODS: Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice. Lewis lung carcinoma cells were injected into C57BI/6 mice. Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose). Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days. Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy. Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells. Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy. RESULTS: In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21). By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone. Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors. Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone. Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone. Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells. Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone. DISCUSSION: The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors. Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent. The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction.

Animals↗