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Biomedical subjects

S Prakash

Publications and source records attributed to S Prakash.

At least 145 records · Page 8Linked to original sources
PubMed1996

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected.

Amino Acid Sequence↗
PubMed1996

Ultraviolet radiation-induced production of superoxide radicals by selected antibiotics.

The ability of certain drugs and chemicals to induce cutaneous phototoxicity and DNA damage has been attributed to free radical formation during photolysis. In this context we have observed that the synergistic action of commonly used antibiotics and ultraviolet radiation (UVR) exhibited strong superoxide radical (O2-) generation potential in the following order: benzylpenicillin > amphotericin > ampicillin > nystatin > spectinomycin > gentamicin. Commercially available penicillin, nystatin, ampicillin and gentamicin also generated O2- under similar conditions. The results suggest that due precaution are necessary to avoid UVR after the intake of photoreactive drugs.

Anti-Bacterial Agents↗
PubMed1996

Microencapsulated genetically engineered E. coli DH5 cells for plasma urea and ammonia removal based on: 1. Column bioreactor and 2. Oral administration in uremic rats.

We report a novel approach for plasma urea and ammonia removal using artificial cells microencapsulated genetically engineered bacteria E. coli DH5 cells. This has been evaluated for use in a column bioreactor for removing plasma urea and ammonia. It has also been evaluated in uremic rats for urea removal by oral administration. In 30 minutes, microencapsulated E. coli DH5 in a column bioreactor in-vitro lowered plasma urea to 10.47 +/- 3.45 mg/dl from 45.85 +/- 2.98 mg/dl and lowered plasma ammonia concentration to 46.00 +/- 4.00 microM from 679 +/- 32 microM/1. The efficiency of this bioreactor for plasma urea and ammonia removal is much higher than any other available methods. Initial plasma urea and ammonia concentration does not affect the plasma urea and ammonia removal efficiency of the column bioreactor. In in-vivo studies of oral administration to uremic rats, both free and encapsulated bacteria both lowered systemic urea from the initial 52.08 (S.D.2.37) mg/dl to 10.58 (S.D.0.85) mg/dl. Unlike free bacteria, microencapsulated bacteria was not retained in the intestine.

Administration, Oral↗
PubMed1996

Development of malathion resistance in Culex quinquefasciatus Say (Diptera: Culicidae).

A malathion resistant colony of C. quinquefasciatus was developed in the laboratory. LC50 and LC90 for larvae were calculated at every generation and the values were 0.3 ppm and 1.13 ppm for first generation and 61.09 ppm, 136.3 ppm for 25th generation respectively. The fold increase in LC50 and LC90 were 2036 and 2726 folds respectively. Cross resistance against propoxur and chlorpyrifos showed 6.64 and 6.52 fold and 600 and 720 fold increase in their LC50 and LC90 values respectively. Triphenyl phosphate (TPP) and piperonyl butoxide (PB) were used as synergists and TPP indicated proportional decrease in LC50 and LC90 values while not much change was observed with PB. No change in biotic potential (larval hatchability, adult emergence and male and female ratio) between susceptible and malathion resistant colonies was observed.

Animals↗
PubMed1996

Assessment of iodine deficiency disorders using the 30 cluster approach in the National Capital Territory of Delhi.

OBJECTIVE: Iodine Deficiency Disorders (IDD) are a major public health problem in India. The National Capital Territory (NCT) of Delhi is a known iodine deficiency endemic area. The Delhi Government banned the sale of non-iodised salt since 1989. The present study was conducted to assess the status of IDD after 7 years of salt iodisation programme in the state. DESIGN: Cross sectional. METHODOLOGY: The recent indicators recommended by the World Health Organization-United Nations Childrens Fund-International Council for the Control of Iodine Deficiency Disorders (WHO-UNICEF-ICCIDD) were utilized for the assessment of IDD. A total of 30 clusters were selected using population proportionate to size cluster sampling procedure. In each identified cluster, one primary school was selected using random sampling. A total of 6911 school children in the age group of 8-10 years were included for the study. RESULTS: The total goiter prevalence rate was 8.6% while 2.1, 8.4, 17.6 and 71.9% of the children had urinary iodine excretion levels of < 2, 2-4.9, 5-9.9 and 10 and above mcg/dl, respectively. The median urinary iodine excretion was 17 mcg/dl. Of the 1854 salt samples analyzed, salt with a nil iodine content was consumed only by 1.4% of the beneficiaries. Forty one per cent of families consumed salt with an iodine content of less than 15 ppm. CONCLUSION: IDD continues to be a public-health problem in the NCT of Delhi. There is a need of strengthening the existing monitoring system for the quality of iodised salt.

Child↗
PubMed1995

Structure-specific nuclease activity in yeast nucleotide excision repair protein Rad2.

Saccharomyces cerevisiae Rad2 protein functions in the incision step of the nucleotide excision repair of DNA damaged by ultraviolet light. Rad2 was previously shown to act endonucleolytically on circular single-stranded M13 DNA and also to have a 5'-->3' exonuclease activity (Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1993) Nature 366, 365-368; Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1994) J. Biol. Chem. 269, 31342-31345). Using two different branched DNA structures, pseudo Y and flap, we have determined that Rad2 specifically cleaves the 5'-overhanging single strand in these DNAs. Rad2 nuclease is more active on the flap structure than on the pseudo Y structure. Rad2 also acts on a bubble structure that contains an unpaired region of 14 nucleotides, but with a lower efficiency than on the pseudo Y or flap structure. The incision points occur at and around the single strand-duplex junction in the three classes of DNA structures.

Base Sequence↗
PubMed1995

Lethality in yeast of trichothiodystrophy (TTD) mutations in the human xeroderma pigmentosum group D gene. Implications for transcriptional defect in TTD.

Mutations in the human XPD gene result in a defect in nucleotide excision repair of ultraviolet damaged DNA and cause the cancer-prone syndrome xeroderma pigmentosum (XP). Besides XP, mutations in XPD can cause another seemingly unrelated syndrome, trichothiodystrophy (TTD), characterized by sulfur-deficient brittle hair, ichthyosis, and physical and mental retardation. To ascertain the underlying defect responsible for TTD, we have expressed the TTD mutant proteins in the yeast Saccharomyces cerevisiae and determined if these mutations can rescue the inviability of a rad3 null mutation. RAD3, the S. cerevisiae counterpart of XPD, is required for nucleotide excision repair and also has an essential role in RNA polymerase II transcription. Expression of the wild type XPD protein or the XPD Arg-48 protein carrying a mutation in the DNA helicase domain restores viability to the rad3 null mutation. Interestingly, the XPD variants containing TTD mutations fail to complement the lethality of the rad3 null mutation, strongly suggesting that TTD mutations impair the ability of XPD protein to function normally in RNA polymerase II transcription. From our studies, we conclude that XPD DNA helicase activity is not essential for transcription and infer that TTD mutations in XPD result in a defect in transcription.

Amino Acid Sequence↗
PubMed1995

Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.

Simple repetitive DNA sequences are unstable in human colorectal cancers and a variety of other cancers. Mutations in the DNA mismatch repair genes MSH2, MLH1, and PMS1 result in elevated rates of spontaneous mutation and cause a marked increase in the instability of simple repeats. Compared with the wild type, a null mutation in the yeast RTH1 gene, which encodes a 5' to 3' exonuclease, was shown to increase the rate of instability of simple repetitive DNA by as much as 280 times and to increase the spontaneous mutation rate by 30 times. Epistasis analyses were consistent with the hypothesis that this RTH1-encoded nuclease has a role in the MSH2-MLH-1-PMS1 mismatch repair pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH.

Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5'-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.

2-Acetylaminofluorene↗
PubMed1995

Yeast DNA repair protein RAD23 promotes complex formation between transcription factor TFIIH and DNA damage recognition factor RAD14.

In Saccharomyces cerevisiae, the multisubunit RNA polymerase II general transcription factor TFIIH is indispensable for transcription initiation and some of its subunits are known to be required for nucleotide excision repair (NER) of DNA damaged by ultraviolet light. RAD3, a subunit of TFIIH, binds UV-damaged DNA in an ATP-dependent manner. It has, however, remained unclear how TFIIH is assembled with the other damage recognition component RAD14. Here, we demonstrate a higher order complex consisting of TFIIH, RAD14, and another NER protein RAD23, and complex formation between TFIIH and RAD14 is facilitated by the RAD23 protein.

DNA Damage↗
PubMed1995

Cloning and analysis of murine cDNA that encodes a fibrogenic lymphokine, fibrosin.

Tissue fibrosis that complicates chronic inflammation can be a cause of serious morbidity. The molecular links between inflammation and fibrosis appear to be a variety of proteins produced by activated chronic inflammatory cells. Collectively, these fibrogenic cytokines promote the growth of fibroblasts and the production of extracellular matrix that are the characteristic features of fibrotic tissue. In an attempt to clone cDNA for a fibrogenic lymphokine that we had isolated, we transfected COS-7 cells with a cDNA library derived from concanavalin A-stimulated lymphocyte line CDC25. Conditioned medium from the transfected COS-7 cells but not from sham-transfected cells stimulates fibroblast proliferation in vitro. We used heterologous expression in COS-7 cells of pools of CDC25 cDNA and screening for biological activity in conditioned medium to enrich for the cDNA clone(s) that encodes this activity. With this strategy of sib selection we isolated clone 2B3. The culture supernatants of 2B3-transfected COS-7 cells exert maximum growth-stimulating effects on fibroblasts at a dilution of 1:20,000. The isolated cDNA has one open reading frame (216 nucleotides) that has no significant homology with nucleotide sequences that encode other proteins. A synthetic peptide constructed from the deduced amino acid sequence is biologically active in picomolar concentrations, even though it may represent only a portion of the native fibrosin. This lymphokine, which we designate fibrosin, may play a role in regulating fibrogenesis in certain chronic inflammatory diseases.

Amino Acid Sequence↗
PubMed1995

Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems.

A 5'- to 3'-exonuclease of about 45 kDa has been purified from various mammalian sources and shown to be required for the completion of lagging strand synthesis in reconstituted DNA replication systems. RTH1 encodes the yeast Saccharomyces cerevisiae counterpart of the mammalian enzyme. To determine the in vivo biological role of RTH1-encoded 5'- to 3'-exonuclease, we have examined the effects of an rth1 delta mutation on various cellular processes. rth1 delta mutants grow poorly at 30 degrees C, and a cessation in growth occurs upon transfer of the mutant to 37 degrees C. At the restrictive temperature, the rth1 delta mutant exhibits a terminal cell cycle morphology similar to that of mutants defective in DNA replication, and levels of spontaneous mitotic recombination are elevated in the rth1 delta mutant even at the permissive temperature. The rth1 delta mutation does not affect UV or gamma-ray sensitivity but enhances sensitivity to the alkylating agent methyl methanesulfonate. The role of RTH1 in DNA replication and in repair of alkylation damage is discussed.

DNA Damage↗
PubMed1995

Studies on uptake of trivalent and hexavalent chromium by maize (Zea mays).

Pot culture experiments were carried out to study the uptake and translocation of chromium from irrigation water when supplied in its trivalent and hexavalent states to maize plants grown in soil and sand culture. The uptake of chromium was observed to increase with increase in the concentration for both oxidation states of chromium. For the root, the observed order of uptake was CrIII sand > CrVI sand > CrIII soil > CrVI soil, whereas in the lower shoot, upper shoot and fruit the order was CrVI sand > CrVI soil > CrIII sand > CrIII soil. Significantly high uptake of chromium by roots in the sand culture (CrIII treatment) is attributed to the effect of root exudates and degradation products on the mobilization of CrIII. In aerial parts of the plants a higher uptake was observed when the initial supply was CrVI. The trends observed are explained on the basis of the redox behaviour of chromium.

Chromium↗
PubMed1995

Plant uptake of fluoride in irrigation water by ladyfinger (Abelmorchus esculentus).

Because of suggestions that food is a rich source of fluoride to humans and the absence of permissible and upper limits of fluoride for irrigation water, plant uptake studies were conducted using fluoride-rich irrigation water. Ladyfinger was grown in sand and soil cultures for 18 wk and the accumulation of fluoride in various plant parts was studied. The potential for ingestion of fluoride by humans through this route was also considered. The percentage uptake was greater in sand-cultured plants than in soil-cultured plants. The root accumulates most of the fluoride supplied through irrigation water and the fruit accumulates the least. Up to 120 mg fluoride/litre of irrigation water did not harm the plants. The ingestion of fluoride by humans from plants irrigated with water containing 10 mg fluoride/litre would be 0.20 mg per 100 g ladyfinger.

Fluorides↗