Biomedical subjects

S Seetharam

Publications and source records attributed to S Seetharam.

At least 37 records · Page 2Linked to original sources

PubMed1994

Effect of processing inhibitors on cobalamin (vitamin B12) transcytosis in polarized opossum kidney cells.

The main objectives of the current study were to investigate the effect of tunicamycin and other posttranslational processing inhibitors on the apical brush border expression of intrinsic factor-cobalamin receptor (IFCR) and the apical to basolateral transcytosis of cobalamin (Cbl). Because of the high and selective expression of IFCR in the apical brush border membrane of opossum kidney (OK) cells (K. S. Ramanujam, S. Seetharam, N. Dahms, and B. Seetharam, (1991) J. Biol. Chem. 266, 13135-13140), we have used cultured OK cells to address these issues. When polarized OK cells grown on culture inserts were incubated with tunicamycin, deoxynojirimycin, swainsonine, or cerulenin, the surface binding of the ligand, intrinsic factor-[56Co]Cbl was inhibited by tunicamycin but not by the other inhibitors. However, Cbl transcytosis was inhibited by both tunicamycin and cerulenin but not with deoxynojirimycin or swainsonine. Incubation of cells with tunicamycin decreased the half-life of IFCR from 48 to 24 h, thus causing faster degradation and depletion of the surface receptor. Incubation of cells with cerulenin resulted in the intralysosomal retention of internalized Cbl. Mature receptor labeled with either [35S]methionine or [3H]mannose was sensitive to digestion with both endoglycosidase H and peptide N-glycosidase F and revealed the presence of two or three N-linked oligosaccharides of the high mannose or hybrid type. Metabolic labeling of OK cells with [3H]palmitic acid revealed that IFCR was palmitoylated and the label was sensitive to treatment with hydroxylamine. Based on these results we suggest that IFCR expression in the apical membrane and Cbl transcytosis in polarized OK cells are regulated by core N-glycosylation but not by further processing of the terminal sugars. In addition, we also suggest that the inhibition of Cbl transcytosis by cerulenin is due to inhibition of postinternalization events.

1-Deoxynojirimycin↗

PubMed1994

Polymorphism of human transcobalamin II: substitution of proline and/or glutamine residues by arginine.

The sequence of transcobalamin II (TC II) cDNA amplified from human fibroblast and colon adenocarcinoma (Caco-2) and the electrophoretic mobility of TC II secreted by these cell lines were analyzed to get some insights into the structural basis for the expression of various polymorphic forms of human TC II. Based on relative anodic mobilities of TC II phenotypes expressed in human serum, TC II expressed in the fibroblast cell line studied and Caco-2 cells were assigned as the MX (medium/extremely slow) and S (slow) types, respectively. Nucleotide sequence analysis of TC II cDNA amplified from these cells revealed that residues Arg and Arg, Gln and Arg, and Gln and Pro were present at positions 234 and 259, respectively, in TC II alleles encoding the X, S and M types. Based on these results, we suggest that differences in the anodic mobilities of the various polymorphic forms of TC II such as the X, S and M types are due to charge difference on the protein caused by the replacement of uncharged residues by arginine at positions 234 and/or 259.

Arginine↗

PubMed1994

Expression of transcobalamin II mRNA in human tissues and cultured fibroblasts from normal and transcobalamin II-deficient patients.

Transcobalamin II (TCII) is an important plasma transporter of cobalamin (Cbl; vitamin B12). In the present study, TCII gene expression in human and rat tissues and in the fibroblasts of patients with TCII deficiency was investigated. Northern-blot analyses revealed expression of TCII mRNA in many human and rat tissues. In humans, this was 14-fold higher in the kidney than in liver, whereas in the rat the levels of expression were similar in the kidney and liver. Southern-blot analysis of genomic DNA from several species revealed sequence similarity in TCII across species. Metabolic labelling and ribonuclease protection assay revealed a 43 kDa TCII protein and a fully protected TCII mRNA band in normal fibroblasts but not in fibroblasts from three TCII-deficient patients. Southern-blot analysis of genomic DNA from all these fibroblasts revealed identical restriction patterns on BamHI, HindIII, KpnI, MspI and EcoRI digestion. On the basis of these results, we suggest that TCII is expressed in multiple tissues, and its level of expression in tissues varies within the same and across species. Furthermore, the TCII deficiency characterized in this study is due to the absence of TCII protein which in turn is due to the absence or extremely low levels of its mRNA and not to detectable gross alterations in the gene structure.

Animals↗

PubMed1994

Identification of two mutant alleles of transcobalamin II in an affected family.

Transcobalamin II (TC II) deficiency is a rare autosomal recessive disease leading to cobalamin (Cbl; Vitamin B12) deficiency characterized by failure to thrive, megaloblastic anemia, impaired immunodefence and neurological manifestations. By means of Southern blotting and sequence analysis of TC II cDNA amplified from fibroblasts of an affected child and his parents, we have identified two mutant TC II alleles, one with a gross deletion and the other with a 4 nucleotide deletion. Both the mutations caused TC II mRNA and protein deficiency and hence defective plasma transport of Cbl and the development of Cbl deficiency in the affected child. The present study has identified molecular defects that cause TC II deficiency and lead to intracellular Cbl deficiency in humans.

Alleles↗

PubMed1993

Regulated expression of intrinsic factor-cobalamin receptor by rat visceral yolk sac and placental membranes.

Intrinsic factor-cobalamin receptor (IFCR) activity in visceral yolk sac and placental membranes is regulated during pregnancy in rats. While the IFCR activity declined in the visceral yolk sac membranes by 15-fold, it rose nearly 20-fold in the placental membranes from fourteen to nineteen days of gestation. The visceral yolk sac membranes revealed a 230 kDa protein that co-migrated with pure rat renal IFCR. This 230 kDa band was also identified as IFCR in both the membranes by immunoblotting with anti-serum to rat renal IFCR. Immunoprecipitation of 35S labeled proteins obtained from in vitro translation using visceral yolk sac mRNA from 14-day pregnant rats, yielded on SDS-PAGE a single band of 220 kDa, while those obtained from 19-day pregnant rats did not. The binding of intrinsic factor-cyano[57Co]cobalamin complex to the visceral yolk sac membranes was inhibited by preincubation of these membranes with anti-serum to rat IFCR but not with anti-serum to rat asialoglycoprotein receptor or mannose or mannan or N-acetylglucosamine. Based on these results, we suggest that the IFCR activity, protein expression and mRNA levels in fetal membranes are regulated during pregnancy and may play an important role in the maternal-fetal transfer of cobalamin.

Animals↗

PubMed1993

Isolation and sequence analysis of variant forms of human transcobalamin II.

Two cDNA clones (1.9 kb and 1.5 kb, respectively) encoding full length human TC II have been isolated from a human endothelial cell cDNA library and sequenced. The differences between the two clones are the length of the 5' end and the 3' end non-coding regions and the codon at position 198 and 219. Both the clones differ from the recently isolated (human endothelial cell) cDNA for TC II (Platica, O., Janecko, R., Quadros, E.V., Regee, A., Romain, R. and Rothenberg, S.P. (1991) J. Biol. Chem. 266, 7860-7863) in codon 259 and 376 and in their calculated pI values. In vitro transcription followed by translation in a reticulocyte lysate system and SDS-PAGE revealed that the isolated cDNA clones encode a protein of 43 kDa. Upon treatment with canine pancreatic microsomes, the molecular mass of the in vitro translated product was reduced to 41.5 kDa, indicating the presence of an approximately 1.5 kDa signal peptide. This translation product was immunoprecipitated with rabbit anti-serum to human TC II and was able to bind to Cbl-Sepharose beads. The amino acid sequence alignment of TC II with that of other Cbl binding proteins (rat intrinsic factor, human transcobalamin I and porcine haptocorrin) revealed only 33% overall homology. However, there were four regions of greater than 80% homology and two regions of about 60% homology. These regions encompass the majority of the hydrophobic areas of the Cbl-binders. Based on these studies, we suggest that structural basis for the expression of different polymorphic forms of TC II may be due to single point mutations and that TC II, like other mammalian Cbl-binders, have evolved from a common ancestral gene. Furthermore, the Cbl-binding functional domain most probably resides in a hydrophobic pocket which is formed by all or some of the six regions of high homology.

Amino Acid Sequence↗

PubMed1993

Intrinsic factor-cobalamin receptor activity in a marsupial, the American opossum (Didelphis virginiana).

1. Significant and specific binding of intrinsic factor-cobalamin occurred in proximal but not in the distal half of the intestine in an adult marsupial, the American opossum. 2. The purified opossum kidney receptor, like rat and canine kidney receptors, revealed a single band of M(r) approximately 230 on SDS-PAGE. However, unlike the rat and canine receptors, the opossum receptor was sensitive to both Endoglycosidase H and peptide-N-glycosidase F. 3. The opossum intrinsic factor-cobalamin receptor demonstrated a ten-fold higher affinity for intrinsic factor-cobalamin complex when the source of IF was from the opossum pancreas, rather than rat stomach. 4. The opossum kidney receptor had low immune-crossreactivity with anti-serum raised to rat and canine kidney receptor. 5. These studies suggest that intrinsic factor-cobalamin receptor expressed in the American opossum, though conserved, appears to be structurally different from the rat and canine receptors.

Animals↗

PubMed1993

Ultraviolet hypermutability of a shuttle vector propagated in xeroderma pigmentosum variant cells.

Patients with the variant form of xeroderma pigmentosum (XP) have clinical XP including a high frequency of skin cancer but, in contrast to the other forms of XP, have normal post-ultraviolet (UV) DNA excision repair and nearly normal post-UV survival. However, like excision repair-deficient XP cells, the XP variant cells are UV hypermutable. We used a UV-treated plasmid shuttle vector, pZ189, to examine the DNA repair defect in lymphoblastoid cells from an XP variant patient, XPPHBE, and a normal control. Plasmid repair, mutagenesis, and replication occur within transfected cells in a process dependent on the cells' repair capacity. With the XP variant cells post-UV, plasmid survival was normal with but there was an abnormally increased post-UV plasmid mutation frequency. Sequence analysis of the mutated plasmids revealed an increased frequency of plasmids with single base substitution mutations with the XP variant cells. As in earlier studies with UV mutagenesis, there was a predominance of G:C-->A:T base substitution mutations with plasmids recovered from both cell lines. The frequency of G:C-->C:G transversions was significantly higher with plasmids recovered from the XP variant cells than from normal cells. The location of mutations in the marker gene was non-random with different mutagenic hotspots found in plasmids recovered from the XP variant cells and from the normal cells. This study suggests that plasmid UV hypermutability in the presence of normal UV survival may be related to the increased UV skin cancer susceptibility of XP variant patients.

Adult↗

PubMed1992

Modulation of ultraviolet light mutational hotspots by cellular stress.

Stressful treatments of cells provoke broad, transient, changes in cellular physiology and gene expression. In addition to these effects, DNA-damaging agents often induce permanent change in the form of mutations. Mutational patterns in target genes typically show hotspots and coldspots, the molecular basis of which appears to lie in the sequence context of the particular site. We determined the mutational pattern in an ultraviolet light-modified (in vitro) marker gene in a shuttle vector passaged through repair deficient (xeroderma pigmentosum) cells and compared it with patterns obtained from cells exposed to stress imposed by a DNA-damaging agent or a calcium ionophore. We found that the mutational hotspot pattern was altered by both stress treatments. We conclude that the cellular environment can influence the probability of mutagenesis at specific sites and propose that some of these effects on mutagenesis are mediated by alterations in cellular calcium levels.

Base Sequence↗

PubMed1992

Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex.

The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.

Animals↗

PubMed1992

Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells.

The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis.

Ammonium Chloride↗

PubMed1992

What is known about the structure and function of the Escherichia coli protein FirA?

The firA gene is essential for growth of Escherichia coli growth and lies in the 4-minute region of the genome. firA encodes the FirA protein which contains 341 amino acids and has an apparent molecular mass of 36 kDa. Genetic evidence suggests that FirA plays a role in transcription since certain firA alleles confer temperature sensitivity for growth and RNA synthesis as well as reversing the rifampin resistance of rifampin-resistant rpoB mutants ('fir' effect). FirA co-immunoprecipitates with RNA polymerase holoenzyme, implying a physical association with the transcriptional machinery, possibly with the beta subunit of RNA polymerase. FirA contains a previously undescribed isoleucine/valine-rich six-amino-acid repeat occurring 14 times within the N-terminal and 12 more times within the C-terminal half of the protein. This repeat can be formulated as [HXXXhZ]n with 'H' representing a large non-polar residue (usually isoleucine), 'h' representing a smaller non-polar residue, 'Z' representing either charged/polar or aromatic residues, XXX representing residues typical of beta turns, and 'n' being equal to the repeat number. We refer to this repeat as an isoleucine patch. Proteins encoded by three E. coli acyltransferases also contain this motif which is roughly positioned in each case, within the amino- and carboxyl termini, as in FirA. When the sequences of these proteins are aligned, a region of poor similarity separates the isoleucine patches. The significance of these repeats remains unknown although we speculate that they play an important structural role in the organization and function of FirA (and other proteins containing isoleucine patches), possibly by acting as homo- or hetero-dimerization interfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Intrinsic factor receptor activity and cobalamin transport in bile duct-ligated rats.

The intrinsic factor (IF)-cobalamin (Cbl) receptor activity in the mucosal homogenates progressively decreased after bile duct ligation in the rat, and 80% of the receptor activity was decreased in 96 h after ligation. The activity was restored to normal values of 5.5-6 pmol of IF-[57Co]Cbl bound/g mucosa when the assays were performed with both conjugated and unconjugated bile acids. When [57Co]Cbl bound to intrinsic factor was orally administered, the tissue levels of [57Co]Cbl were decreased by 75-80% in bile duct-ligated rats. The apical membrane receptor activity was also decreased after bile duct ligation; however, the activity was stimulated twofold by the addition of ileal cytosol and threefold with the addition of both ileal cytosol and taurocholate (1 mM). Enhanced binding of IF-[57Co]Cbl to the apical ileal brush-border membrane occurred with the use of dialyzed ileal cytosol but not with cytosol isolated from duodenal or proximal jejunal mucosa. The enhanced binding obtained with ileal cytosol was abolished after its treatment with trypsin. These results suggest that luminal bile acids optimize the binding of IF-Cbl by the ileal membrane receptor via interactions with a cytosolic factor and thus influence the gastrointestinal absorption of cobalamin.

Animals↗

PubMed1991

Functional expression of transcobalamin II cDNA in Xenopus laevis oocytes.

The products of in vitro transcription of human transcobalamin II (TC II) cDNA when microinjected into Xenopus laevis oocytes yielded a single secretory protein of 43 kDa. The mobility of the 43 kDa band did not change following digestion with peptide N-glycosidase F. [57Co]Cbl bound to the medium was immunoprecipitated with anti-serum to human TC II, but not to other Cbl binders. In addition, the [57Co]Cbl complex also bound to placental microsomes. These results suggest that TC II mRNA transcribed encodes TC II which contains both the Cbl and receptor binding domains. Furthermore, Xenopus oocytes can be used as a screening system to define structural elements important in TC II's secretion and binding reactions.

Animals↗

PubMed1991

Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL.

Pseudomonas exotoxin (PE) is a 66,000 molecular weight protein secreted by Pseudomonas aeruginosa. PE is made up of three domains, and PE40 is a form of PE which lacks domain Ia (amino acids 1-252) and has very low cytotoxicity because it cannot bind to target cells. The sequence Arg-Glu-Asp-Leu-Lys (REDLK) at the carboxyl terminus of Pseudomonas exotoxin has been shown to be important for its cytotoxic activity (Chaudhary, V. K., Jinno, Y., FitzGerald, D. J., and Pastan, I. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 308-312). In this study, we tested the effect of altering the carboxyl sequence of PE from REDLK to the characteristic endoplasmic reticulum retention sequence, KDEL, or to KDEL repeated three times (KDEL)3. We also made similar changes at the carboxyl terminus of two chimeric toxins in which domain I of PE (amino acids 1-252) was either replaced with transforming growth factor alpha (TGF alpha) to make TGF alpha-PE40 or with a single chain antibody (anti-Tac) reacting with the human interleukin 2 receptor to make anti-Tac(Fv)-PE40. Statistical analyses of our results demonstrate that PE and its derivatives ending in KDEL or (KDEL)3 are significantly more active than PE or derivatives ending in REDLK. We have also found that brefeldin A, which is known to perturb the endoplasmic reticulum, inhibits the cytotoxic action of PE. Our results suggest that the altered carboxyl terminus may enable the toxin to interact more efficiently with a cellular component involved in translocation of the toxin to the cytosol.

ADP Ribose Transferases↗

PubMed1991

Synthesis and secretion of cobalamin binding proteins by opossum kidney cells.

Opossum kidney epithelial cells synthesize and secrete two Cobalamin (Cbl) binding proteins of Mr 66,000 and 43,000. When grown on culture inserts, the apical medium contained both these proteins while the basolateral medium contained only the 43 kDa Cbl binder. Colchicine, a microtubule disruptive drug, increased two fold the apical but not the basolateral secretion of the Cbl binding proteins. Although the opossum Cbl binders did not cross react with anti-serum raised to Cbl binders from other species, the identity based on Cbl binding and size suggest that the 66 kDa and 43 kDa proteins are haptocorrin and transcobalamin II.

Animals↗

PubMed1991

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.

Animals↗

PubMed1991

In vitro translation and expression of renal intrinsic factor-cobalamin receptor.

The primary translation product of intrinsic factor (IF)-cobalamin receptor (IFCR) mRNA from rat kidney is a single polypeptide chain of Mr = 215,000. When expressed in Xenopus laevis oocytes the IFCR binding activity is expressed with mRNA of a size between 5 to 7 kb. These results suggest that IFCR mRNA transcripts are present in the renal tissue and encode a single chain, large molecular weight precursor. Furthermore, Xenopus oocytes can be used as a screening system in the expression cloning of the renal IFCR.

Animals↗