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Biomedical subjects

S Prakash

Publications and source records attributed to S Prakash.

At least 181 records · Page 10Linked to original sources
PubMed1993

Purification and characterization of the Saccharomyces cerevisiae RAD1/RAD10 endonuclease.

The Saccharomyces cerevisiae RAD1 and RAD10 genes are required for the incision step of excision repair, and in addition, they function in mitotic recombination. The RAD1 and RAD10 proteins are associated in a tight complex, and genetic studies have indicated that complex formation is essential for the RAD1/RAD10 controlled biological activities. We had previously purified the RAD10 protein to near homogeneity from yeast and shown that it is a DNA-binding protein with a strong preference for single-stranded DNA. In this study, we purify the RAD1 protein to near homogeneity from yeast and show that it also binds single-stranded DNA preferentially and that the RAD1/RAD10 complex possesses an endonuclease activity. We characterize the RAD1/RAD10 endonuclease activity on both single-stranded and double-stranded DNAs, using agarose gel electrophoresis and trichloroacetic acid precipitation. The RAD1/RAD10 nuclease exhibits a much higher level of activity on single-stranded DNA than double-stranded DNA. The susceptibility of double-stranded DNA to nicking by the RAD1/RAD10 enzyme is markedly dependent on the degree of negative superhelicity, such that a 15-fold increase in nicking rate is observed from superhelical state sigma = zero to sigma = -0.08. The enzyme produces 3'-hydroxyl and 5'-phosphate termini on both single- and double-stranded DNAs. We discuss the role of RAD1/RAD10 endonuclease in nucleotide excision repair and in mitotic recombination.

Chromatography, Ion Exchange↗
PubMed1993

Yeast excision repair gene RAD2 encodes a single-stranded DNA endonuclease.

In eukaryotes nucleotide excision repair of DNA damaged by ultraviolet radiation requires several gene products; defects in this process result in the cancer-prone syndrome xeroderma pigmentosum (XP) in humans. The RAD2 gene is one of at least seven genes indispensable for excision repair in the yeast Saccharomyces cerevisiae, and its encoded protein shares remarkable homology with the XP group-G gene product. Here we overproduce the RAD2-encoded protein in S. cerevisiae, purify it to near homogeneity, and show that RAD2 protein in the presence of magnesium degrades circular single-stranded DNA. The RAD2 endonuclease is specific for single-stranded DNA as it does not act on double-stranded DNA. Given the absolute requirement for RAD2 in the incision step of excision repair, our findings directly implicate RAD2 protein and its human homologue XPG protein as a catalytic component that incises the damaged DNA strand during excision repair. Furthermore, our results indicate that eukaryotes probably employ two distinct endonuclease activities to mediate the dual incision at the damage site.

DNA Repair↗
PubMed1993

Human xeroderma pigmentosum group D gene encodes a DNA helicase.

Xeroderma pigmentosum (XP), a genetically heterogeneous human disease, results from a defect in nucleotide excision repair of ultraviolet-damaged DNA. XP patients are extremely sensitive to sunlight and suffer from a high incidence of skin cancers. Cell fusion studies have identified seven XP complementation groups, A-G. Group D is of particular interest as mutations in this gene can also cause Cockayne's syndrome and trichothiodystrophy. The XPD gene was initially named ERCC2 (excision repair cross complementing) as it was cloned using human DNA to complement the ultraviolet sensitivity of a rodent cell line. We have purified the XPD protein to near homogeneity and show that it possesses single-stranded DNA-dependent ATPase and DNA helicase activities. We tested whether XPD can substitute for its yeast counterpart RAD3, which is essential for excision repair and for cell viability. Expression of the XPD gene in Saccharomyces cerevisiae can complement the lethality defect of a mutation in the RAD3 gene, suggesting that XPD is an essential gene in humans.

Adenosine Triphosphatases↗
PubMed1993

Studies on the leaching of fluoride in tea infusions.

In order to assess the levels of fluoride ingestion through intake of tea, studies were conducted with four different brands of tea leaves commonly available in the Indian market. Four most prevalent methods for the preparation of tea with various contact times (2,4,6,8 and 10 min) of tea leaves with water show that: (a) leaching of fluoride is least in case of leaf tea as compared to powdered tea (F levels increasing with decreasing grain size); (b) leaching of fluoride reaches a maximum after a contact of about 6 min; (c) there is no difference between levels of fluoride with or without addition of milk in the English style where tea leaves are not boiled, while for the Indian style, addition of milk and subsequent boiling resulted in reduction of fluoride levels and (d) ingestion of fluoride per cup of tea ranged from 1.55 mg/l to 3.21 mg/l amounting to an intake per day per person of fluoride between 0.3 to 1.9 mg.

Fluorides↗
PubMed1993

Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA.

Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A-G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. We have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. We also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair.

Amino Acid Sequence↗
PubMed1993

The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II.

The RAD25 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and, in addition, is essential for viability. RAD25 shares a high degree of homology with the human ERCC3/XPBC-encoded protein, and the yeast and human proteins resemble one another in containing the conserved ATPase/DNA helicase sequence motifs. To determine the nature of the essential role of RAD25, we have isolated a recessive temperature-sensitive conditional lethal mutation of the gene and have examined its effect on transcription. Upon shift to the nonpermissive temperature, the rad25 temperature-sensitive (ts) mutant stops growth rapidly and shows a large decrease in the synthesis of poly(A)+ RNA. Transcription of a large number of yeast genes, including HIS3, TRP3, STE2, MET19, RAD23, CDC9, and ACT1 is inhibited at the restrictive temperature in the rad25 ts mutant, and the galactose-inducible synthesis of GAL7 and GAL10 mRNAs is also severely affected by the loss of RAD25 activity. These findings implicate a general requirement of RAD25 in RNA polymerase II transcription.

Amino Acid Sequence↗
PubMed1993

The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation.

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for DNA repair, damage-induced mutagenesis, and sporulation. In addition, RAD6 mediates the multiubiquitination and degradation of amino-end rule protein substrates. The structure and function of RAD6 have been remarkably conserved during eukaryotic evolution. Here, we examine the role of the extremely conserved amino terminus, which has remained almost invariant among RAD6 homologs from yeast to human. We show that RAD6 is concentrated in the nucleus and that the amino-terminal deletion mutation, rad6 delta 1-9, does not alter the location of the protein. The amino-terminal domain, however, is essential for the multiubiquitination and degradation of amino-end rule substrates. In the rad6 delta 1-9 mutant, beta-galactosidase proteins bearing destabilizing amino-terminal residues become long lived, and purified rad6 delta 1-9 protein is ineffective in ubiquitin-protein ligase (E3)-dependent protein degradation in the proteolytic system derived from rabbit reticulocytes. The amino terminus is required for physical interaction of RAD6 with the yeast UBR1-encoded E3 enzyme, as the rad6 delta 1-9 protein is defective in this respect. The rad6 delta 1-9 mutant is defective in sporulation, shows reduced efficiency of DNA repair, but is proficient in UV mutagenesis. E3-dependent protein degradation by RAD6 could be essential for sporulation and could affect the efficiency of DNA repair.

Amino Acid Sequence↗
PubMed1993

Fibroblast-stimulating factor 1, a novel lymphokine produced in schistosomal egg granulomas, stimulates liver fat-storing cells in vitro.

We report that the novel lymphokine fibroblast-stimulating factor 1, produced within hepatic schistosomal egg granulomas, stimulates liver fat-storing cells (FSC) in vitro to proliferate and express fibronectin genes. Because FSC are critical in hepatic fibrogenesis generally, we propose that in vivo stimulation of FSC by fibroblast-stimulating factor 1 may be an important step in schistosomal liver fibrosis.

Adipose Tissue↗
PubMed1993

The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function.

In eukaryotes, the posttranslational conjugation of ubiquitin to various cellular proteins marks them for degradation. Interestingly, several proteins have been reported to contain ubiquitin-like (ub-like) domains that are in fact specified by the DNA coding sequences of the proteins. The biological role of the ub-like domain in these proteins is not known; however, it has been proposed that this domain functions as a degradation signal rendering the proteins unstable. Here, we report that the product of the Saccharomyces cerevisiae RAD23 gene, which is involved in excision repair of UV-damaged DNA, bears a ub-like domain at its amino terminus. This finding has presented an opportunity to define the functional significance of this domain. We show that deletion of the ub-like domain impairs the DNA repair function of RAD23 and that this domain can be functionally substituted by the authentic ubiquitin sequence. Surprisingly, RAD23 is highly stable, and the studies reported herein indicate that its ub-like domain does not mediate protein degradation. Thus, in RAD23 at least, the ub-like domain affects protein function in a nonproteolytic manner.

Amino Acid Sequence↗
PubMed1993

Marrow histomorphology and clinical staging of chronic lymphocytic leukemia--a rare disease in India: experience with 26 cases.

Chronic Lymphocytic Leukemia is a relatively uncommon hemopoietic malignancy in the Indian subcontinent. We have made an attempt to correlate the morphology of the marrow with staging and clinical course of the disease in 26 cases. Four out of 6 cases in Stage A showed a nodular/interstitial marrow pattern, while 18 out of 20 cases of stage B and C demonstrated a mixed/diffuse involvement of marrow. Cases showing a nodular/interstitial pattern had a relatively benign clinical course even without chemotherapy, while patients with diffuse/mixed marrow pattern required chemotherapy. Trephine histological pattern was found to be a good prognosticator and was useful in segregating cases requiring chemotherapy from those which do not.

Aged↗
PubMed1993

Genetically engineered E. coli cells containing K. aerogenes gene, microencapsulated in artificial cells for urea and ammonia removal.

Microencapsulated genetically engineered E. coli cells can efficiently remove urea without any increase in the ammonia levels in the medium. A 100 mg. alginate encapsulated bacteria rapidly reduces urea in a 100 ml. solution. The original urea concentration 100.00 +/- 1.00 mg./dl. fell to 1.55 +/- 0.13 mg./dl. in 30 minutes. There was no increase in the ammonia in the reaction medium. Extrapolated results shows that urea depletion capacity of encapsulated bacteria is sufficient to remove urea during kidney failure. Using single pool model, 40 gm. of encapsulated genetically engineered E. coli can lower urea (100 mg./dl.) in 40 litres of the body water to 1.60 mg./dl. within 30 minutes. Also, 40.00 gm. bacteria can lower ammonia (758.00 microM/l), in 40 litres of body water, to 90.42 microM/l in 20 minutes. Further studies will be required for multi-compartmental models in the physiological conditions.

Ammonia↗
PubMed1992

RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability.

Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M(r) 95,356). The RAD25 (SSL2)- and XPBC-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UV sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys392 residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD25 (SSL2) ATPase/DNA helicase activity in viability.

Amino Acid Sequence↗
PubMed1992

Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for excision repair of ultraviolet light-damaged DNA, and they also function in a mitotic recombination pathway that is distinct from the double-strand-break recombination pathway controlled by RAD52. Here, we show that the RAD1 and RAD10 proteins are complexed with each other in vivo. Immunoprecipitation of yeast cell extracts with either anti-RAD1 antibody or anti-RAD10 antibody coprecipitated quantitative amounts of both RAD1 and RAD10 proteins. The level of coprecipitable RAD1 and RAD10 increased when both proteins were overproduced together, but not if only one of the proteins was overproduced. The RAD1/RAD10 complex is highly stable, being refractory to 1 M NaCl and to low concentrations of SDS. By hydroxylamine mutagenesis, we have identified a rad1 mutant allele whose encoded protein fails to complex with RAD10. The interaction-defective rad1 mutant resembles the rad1 or rad10 null mutant in defective DNA repair and recombination, implying that complex formation is essential for the expression of biological activities controlled by RAD1 and RAD10.

DNA Repair↗
PubMed1992

Fibroblast stimulation in schistosomiasis. XII. Identification of CD4+ lymphocytes within schistosomal egg granulomas as a source of an apparently novel fibroblast growth factor (FsF-1).

Granulomas that form around Schistosoma mansoni eggs deposited in the liver secrete a variety of fibrogenic factors that may provide a molecular link between chronic inflammation and hepatic fibrogenesis in schistosomiasis. We recently isolated from conditioned medium of egg granuloma cultures a approximately equal to 60-kDa heparin-binding growth factor for fibroblasts. Because this protein is distinct from other defined heparin-binding growth factors, we designated it "fibroblast stimulating factor-1" (FsF-1). We now report that FsF-1 is a lymphokine. We prepared IgG antibody against purified FsF-1 and determined that it did not cross-react with a variety of growth factors or recombinant interleukins. Using two-color flow cytometry of dissociated granuloma cell suspensions, we observed that approximately 20% to 25% of granuloma CD4+ lymphocytes express surface FsF-1. We isolated CD4+ granuloma lymphocytes by FACS and observed that these cells spontaneously secrete into culture supernatant a fibroblast mitogen that is neutralized by anti-FsF-1 antibody. Furthermore, anti-FsF-1 can specifically immunoprecipitate a metabolically labeled protein produced by the granuloma CD4+ lymphocytes. The labeled protein has the same apparent molecular mass (approximately equal to 60 kDa) as FsF-1 purified from granuloma culture supernatants. These findings define CD4+ lymphocytes as a source of FsF-1. Because FsF-1 has biologic and chemical features distinct from most other defined lymphokines and from other heparin-binding growth factors, FsF-1 appears to be a novel lymphokine.

Animals↗