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Biomedical subjects

S Prakash

Publications and source records attributed to S Prakash.

At least 127 records · Page 7Linked to original sources
PubMed1996

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro.

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.

Animals↗
PubMed1996

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair.

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

Cockayne Syndrome↗
PubMed1996

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.

Centrifugation, Density Gradient↗
PubMed1996

Binding of insertion/deletion DNA mismatches by the heterodimer of yeast mismatch repair proteins MSH2 and MSH3.

DNA-mismatch repair removes mismatches from the newly replicated DNA strand. In humans, mutations in the mismatch repair genes hMSH2, hMLH1, hPMS1 and hPMS2 result in hereditary non-polyposis colorectal cancer (HNPCC) [1-8]. The hMSH2 (MSH for MutS homologue) protein forms a complex with a 160 kDa protein, and this heterodimer, hMutSalpha, has high affinity for a G/T mismatch [9,10]. Cell lines in which the 160 kDa subunit of hMutSalpha is mutated are specifically defective in the repair of base-base and single-nucleotide insertion/deletion mismatches [9,11]. Genetic studies in S. cerevisiae have suggested that MSH2 functions with either MSH3 or MSH6 in mismatch repair, and, in the absence of the latter two genes, MSH2 is inactive [12,13]. MSH6 encodes the yeast counterpart of the 160 kDa subunit of hMutSalpha [12,13]. As in humans, yeast MSH6 forms a complex with MSH2, and the MSH2-MSH6 heterodimer binds a G/T mismatch [14]. Here, we find that MSH2 and MSH3 form another stable heterodimer, and we purify this heterodimer to near homogeneity. We show that MSH2-MSH3 has low affinity for a G/T mismatch but binds to insertion/deletion mismatches with high specificity, unlike MSH2-MSH6.

Base Sequence↗
PubMed1996

RAD26, the yeast homolog of human Cockayne's syndrome group B gene, encodes a DNA-dependent ATPase.

Cells from Cockayne's syndrome (CS) patients are sensitive to ultraviolet light and defective in preferential repair of the transcribed DNA strand. CS patients suffer from complex clinical symptoms, including severe growth retardation, neurological degeneration, mental retardation, and cachexia. Two CS complementation groups, CSA and CSB, have been identified so far. RAD26 encodes the yeast counterpart of the CSB gene. Here, we purify Rad26 protein to near homogeneity from yeast cells and show that it is a DNA-dependent ATPase. In contrast to the Mfd protein that functions in transcription-coupled repair in Escherichia coli, and which is a weak and DNA independent ATPase, Rad26 is a much more active ATPase, with a strict dependence on DNA. The possible role of Rad26 ATPase in the displacement of stalled RNA polymerase II from the site of the DNA lesion and in the subsequent recruitment of a DNA repair component is discussed.

Adenosine Triphosphatases↗
PubMed1996

An affinity of human replication protein A for ultraviolet-damaged DNA.

Replication protein A (RPA), a heterotrimeric protein of 70-, 32-, and 14-kDa subunits, is an essential factor for DNA replication. Biochemical studies with human and yeast RPA have indicated that it is a DNA-binding protein that has higher affinity for single-stranded DNA. Interestingly, in vitro nucleotide excision repair studies with purified protein components have shown an absolute requirement for RPA in the incision of UV-damaged DNA. Here we use a mobility shift assay to demonstrate that human RPA binds a UV damaged duplex DNA fragment preferentially. Complex formation between RPA and the UV-irradiated DNA is not affected by prior enzymatic photo-reactivation of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct. We also show that Mg2+ in the millimolar range is required for preferential binding of RPA to damaged DNA. These findings identify a novel property of RPA and implicate RPA in damage recognition during the incision of UV-damaged DNA.

DNA↗
PubMed1996

Reconstitution of TFIIH and requirement of its DNA helicase subunits, Rad3 and Rad25, in the incision step of nucleotide excision repair.

Yeast TFIIH is composed of six subunits: Rad3, Rad25, TFB1, SSL1, p55, and p38. In addition to TFIIH, we have purified a subassembly of the factor that lacks Rad3 and Rad25 and which we refer to as TFIIHi. In the in vitro nucleotide excision repair (NER) system that consists entirely of purified proteins, we show that neither TFIIHi nor a mixture of purified Rad3 and Rad25 proteins is active in NER but that the combination of TFIIHi with Rad3 and Rad25 promotes the incision of UV-damaged DNA. These results provide the first evidence for a direct requirement of Rad3, Rad25, and of one or more of the TFIIHi subunits in the incision step of NER. The NER efficacy of TFIIH is greatly diminished or abolished upon substitution of Rad3 with the rad3 Arg-48 mutant protein or Rad25 with the rad25 Arg-392 mutant protein, respectively, thus indicating a role of the Rad3 and Rad25 DNA helicase functions in the incision of damaged DNA. Our results further indicate that the carboxyl-terminal domain kinase (CTD) TFIIK is dispensable for the incision of damaged DNA in vitro. These studies reveal the differential requirement of Rad3 DNA helicase and CTD kinase activities in damage-specific incision versus RNA polymerase II transcription.

Adenosine Triphosphatases↗
PubMed1996

Molecular cloning and tissue distribution of keratocan. Bovine corneal keratan sulfate proteoglycan 37A.

Previous studies showed that the keratan sulfate-containing proteoglycans of bovine corneal stroma contain three unique core proteins designated 37A, 37B, and 25 (Funderburgh, J. L., Funderburgh, M. L., Mann, M. M., and Conrad, G. W. (1991) J. Biol. Chem. 266, 14226-14231). Degenerate oligonucleotides designed from amino acid sequences of the 37A protein were used to screen a cDNA expression library from cultured bovine keratocytes. A cDNA clone coding for keratocan, a 37A protein, was isolated and sequenced. The deduced keratocan amino acid sequence is unique but related to two other keratan sulfate-containing proteins, lumican (the 37B core protein) and fibromodulin. These three proteins share approximately 35% amino acid identity and a number of conserved structural features. Northern hybridization and immunoblotting of tissue extracts found keratocan distribution to be more limited than that of lumican or fibromodulin. Keratocan is abundant in cornea and sclera and detected in much lesser amounts in skin, ligament, cartilage, artery, and striated muscles. Only in cornea was keratocan found to contain large, sulfated keratan sulfate chains. Keratocan, like lumican, is a core protein of a major corneal proteoglycan but is present in non-corneal tissues primarily as a nonsulfated glycoprotein.

Amino Acid Sequence↗
PubMed1996

Nucleotide excision repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome.

In yeast and humans, nucleotide excision repair (NER) of ultraviolet (UV)-damaged DNA requires a large number of highly conserved protein factors, which include the multisubunit RNA polymerase II transcription factor TFIIH. Here, we examine whether NER occurs by sequential assembly of different repair factors at the site of DNA damage or by the placement there of a "preformed" repairosome containing TFIIH and all the other essential NER factors. Contrary to the recent report (Svejstrup, J. Q., Wang, Z., Feaver, W. J., Wu, X., Bushnell, D. A., Donahue, T. F., Friedberg, E. C., and Kornberg, R. D. (1995) Cell 80, 21-28), our results provide no evidence for a pre-assembled repairosome; instead, they support the sequential assembly model. By several independent criteria, including co-purification, immunoprecipitation, and gel filtration of homogeneous proteins, we show that the damage recognition factor Rad14 exists in a ternary complex with the Rad1-Rad10 nuclease. We also find that Rad14 interacts directly with Rad1, but only slightly with Rad10, and that it interacts with the Rad1-Rad10 complex much more efficiently than with Rad1 alone. In the reconstituted NER system, a higher level of incision of UV-damaged DNA is achieved with the Rad1-Rad10-Rad14 complex, which we designate as nucleotide excision repair factor-1, NEF-1.

DNA Damage↗
PubMed1996

Requirement of the yeast MSH3 and MSH6 genes for MSH2-dependent genomic stability.

Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2-dependent mismatch repair.

Amino Acid Sequence↗
PubMed1996

Cyclic and acyclic polyamines as chelators of rhenium-186 and rhenium-188 for therapeutic use.

Several polyamine ligands L1-L7 were assessed as chelators for rhenium-188 (and by analogy, rhenium-186) for incorporation into the design of radiopharmaceuticals for targeted radiotherapy. Both ease of synthesis of the complexes and their kinetic stability in human serum were examined. Chelation of Re-188 by stannous reduction of perrhenate in the presence of acyclic ligands such as L1 and L2 (L1 = ethylenediamine, L2 = 1,4,8,11-tetraazaundecane) proceeded in acceptable yield (50-90%) under aqueous conditions (pH 11; 20-100 degrees C, 30 min) in a single step. In contrast, synthesis of complexes of the cyclic ligands such as L6 (L6 = 1,4,8,11-tetraazacyclotetradecane, cyclam) in acceptable yield (> 50%) required more involved procedures including use of nonaqueous solvents. The chelates were unambiguously identified as the cationic trans-dioxorhenium(V) tetrakis(amino) complexes, by chromatographic comparison with spectroscopically characterised nonradioactive samples. The complexes of tetradentate ligands L2 and L6 showed no evidence of degradation on incubation for up to 24 h in human serum. The complex of L1 degraded by less than 3% under these conditions. These preliminary studies indicate that the acyclic tetradentate ligands offer an appropriate compromise between biological stability and ease of synthesis, and they have potential as chelators for rhenium in radiopharmaceuticals.

Chelating Agents↗
PubMed1996

Study of mercury-selenium (Hg-Se) interactions and their impact on Hg uptake by the radish (Raphanus sativus) plant.

Pot culture experiments were conducted to study the effects of selenite and selenate treatment (0.5-6.0 microg/ml) on the uptake and translocation of root-absorbed mercury (Hg) in radish plants irrigated with 2 and 5 microg/ml Hg in sand and soil culture. Statistically significant reductions in mercury uptake with increasing concentrations of selenium (Se) were observed. Both forms of selenium (selenite and selenate) were equally effective in reducing the mercury burden of the plant. The observed reduction in plant uptake of mercury is explained by the formation of an HgSe insoluble complex in the soil-root environment. No significant difference (P > 0.05) in dry matter yields with the various selenium treatments was found, suggesting that no selenium toxicity or salt injury occurred in the plants.

Drug Interactions↗
PubMed1996

Microencapsulated genetically engineered live E. coli DH5 cells administered orally to maintain normal plasma urea level in uremic rats.

Safety concerns about introducing genetically engineered cells into the body have prevented their use in medical treatments. To solve this problem, we prepared polymeric membrane artificial cells (semipermeable microcapsules) containing genetically engineered live cells from the bacteria Escherichia coli DH5. When given orally, the cells remain at all times in the microcapsules and are finally excreted in the stool. During their passage through the intestine, small molecules like urea diffuse rapidly into the microcapsules and are acted on by the genetically engineered cells. This lowers the high plasma urea level to normal in uremic rats with induced kidney failure, and has exciting implications for the use of this and many other types of genetically engineered cells in a number of medical applications.

Administration, Oral↗