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Biomedical subjects

S Prakash

Publications and source records attributed to S Prakash.

At least 19 recordsLinked to original sources
PubMed1999

Synergistic interaction between yeast nucleotide excision repair factors NEF2 and NEF4 in the binding of ultraviolet-damaged DNA.

Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA. Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4. While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction. Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo. Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP. We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA. Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4.

Adenosine Triphosphatases↗
PubMed1999

hRAD30 mutations in the variant form of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is an autosomal recessive disease characterized by a high incidence of skin cancers. Yeast RAD30 encodes a DNA polymerase involved in the error-free bypass of ultraviolet (UV) damage. Here it is shown that XP variant (XP-V) cell lines harbor nonsense or frameshift mutations in hRAD30, the human counterpart of yeast RAD30. Of the eight mutations identified, seven would result in a severely truncated hRad30 protein. These results indicate that defects in hRAD30 cause XP-V, and they suggest that error-free replication of UV lesions by hRad30 plays an important role in minimizing the incidence of sunlight-induced skin cancers.

Alleles↗
PubMed1999

Requirement of DNA polymerase activity of yeast Rad30 protein for its biological function.

The RAD30 gene of Saccharomyces cerevisiae encodes a DNA polymerase, Poleta. The Rad30 protein shares homology with the yeast Rev1 and the Escherichia coli DinB and UmuC proteins. Although these proteins contain several highly conserved motifs, only Rad30 has been shown to possess a DNA polymerase activity. To determine whether the DNA polymerase activity of Rad30 was essential for its biological function, we made a mutation in the highly conserved SIDE sequence in Rad30, in which the aspartate and glutamate residues have each been changed to alanine. The mutant Rad30 protein lacks the DNA polymerase activity, and the mutant gene does not complement the rad30Delta mutation. These findings indicate that DNA polymerase activity is indispensable for the biological function of RAD30.

Amino Acid Sequence↗
PubMed1999

Proteasome inhibitors: a novel class of potent and effective antitumor agents.

The ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth. Dysregulating the degradation of such proteins should have profound effects on tumor growth and cause cells to undergo apoptosis. To test this hypothesis, we developed a novel series of proteasome inhibitors, exemplified by PS-341, which we describe here. As determined by the National Cancer Institute in vitro screen, PS-341 has substantial cytotoxicity against a broad range of human tumor cells, including prostate cancer cell lines. The PC-3 prostate cell line was, therefore, chosen to further examine the antitumor activity of PS-341. In vitro, PS-341 elicits proteasome inhibition, leading to an increase in the intracellular levels of specific proteins, including the cyclin-dependent kinase inhibitor, p21. Moreover, exposure of such cells to PS-341 caused them to accumulate in the G2-M phase of the cell cycle and subsequently undergo apoptosis, as indicated by nuclear condensation and poly(ADP-ribose) polymerase cleavage. Following weekly i.v. treatment of PS-341 to mice bearing the PC-3 tumor, a significant decrease (60%) in tumor burden was observed in vivo. Direct injection of PS-341 into the tumor also caused a substantial (70%) decrease in tumor volume with 40% of the drug-treated mice having no detectable tumors at the end of the study. Studies also revealed that i.v. administration of PS-341 resulted in a rapid and widespread distribution of PS-341, with highest levels identified in the liver and gastrointestinal tract and lowest levels in the skin and muscle. Modest levels were found in the prostate, whereas there was no apparent penetration of the central nervous system. An assay to follow the biological activity of the PS-341 was established and used to determine temporal drug activity as well as its ability to penetrate tissues. As such, PS-341 was shown to penetrate PC-3 tumors and inhibit intracellular proteasome activity 1.0 h after i.v. dosing. These data illustrate that PS-341 not only reaches its biological target but has a direct effect on its biochemical target, the proteasome. Importantly, the data show that inhibition of this target site by PS-341 results in reduced tumor growth in murine tumor models. Together, the results highlight that the proteasome is a novel biochemical target and that inhibitors such as PS-341 represent a unique class of antitumor agents. PS-341 is currently under clinical evaluation for advanced cancers.

Algorithms↗
PubMed1999

Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Poleta.

The RAD30 gene of the yeast Saccharomyces cerevisiae is required for the error-free postreplicational repair of DNA that has been damaged by ultraviolet irradiation. Here, RAD30 is shown to encode a DNA polymerase that can replicate efficiently past a thymine-thymine cis-syn cyclobutane dimer, a lesion that normally blocks DNA polymerases. When incubated in vitro with all four nucleotides, Rad30 incorporates two adenines opposite the thymine-thymine dimer. Rad30 is the seventh eukaryotic DNA polymerase to be described and hence is named DNA polymerase eta.

Adenine↗
PubMed1999

Columnar and metaplastic cells in vault smears: cytologic and colposcopic study.

Vault smears (n = 250) were examined as routine follow up of hysterectomized individuals. Although the majority of these smears were normal, 14 of them revealed the presence of columnar cells or metaplastic cells. None of these patients had any known cause which could account for the presence of these cells in a post-hysterectomy smear. The possible explanations could be (i) the common mullerian origin of the upper vaginal mucosal cells, (ii) exfoliated reparative squamous parabasal and basal cells mimicking columnar cells, (iii) goblet cell metaplasia in atrophic vaginal epithelium.

Adult↗
PubMed1999

Vanadium: a review of its potential role in the fight against diabetes.

The potential role of vanadium in human health is described as a building material of bones and teeth. However, another very interesting and promising application for vanadium in human health emerges from recent studies that evaluated the role of vanadium in the management of diabetes. Vanadium is present in a variety of foods that we commonly eat. Skim milk, lobster, vegetable oils, many vegetables, grains and cereals are rich source of vanadium (>1 ppm). Fruits, meats, fish, butter, cheese, and beverages are relatively poor sources of vanadium. The daily dietary intake in humans has been estimated to vary from 10 microg to 2 mg of elemental vanadium, depending on the environmental sources of this mineral in the air, water, and food of the particular region tested. In animals, vanadium has been shown essential (1-10 microg vanadium per gram of diet). There is only circumstantial evidence that vanadium is essential for humans. However, in doses ranging from 0.083 mmol/d to 0.42 mmol/d, vanadium has shown therapeutic potential in clinical studies with patients of both insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM) type. Although vanadium has a significant biological potential, it has a poor therapeutic index, and attempts have been made to reduce the dose of vanadium required for therapeutic effectiveness. Organic forms of vanadium, as opposed to the inorganic sulfate salt of vanadium, are recognized as safer, more absorbable, and able to deliver a therapeutic effect up to 50% greater than the inorganic forms. The goal is to provide vanadium with better gastrointestinal absorption, and in a form that is best able to produce the desired biological effects. As a result, numerous organic complexes of vanadium have been developed including bis(maltolato)oxovanadium (BMOV), bis(cysteinamide N-octyl)oxovanadium known as Naglivan, bis(pyrrolidine-N-carbodithioato)oxovanadium, vanadyl-cysteine methyl ester, and bis-glycinato oxovanadium (BGOV). The health benefits of vanadium and the safety and efficacy of the available vanadium supplements are discussed in this review.

Diabetes Mellitus, Type 1↗
PubMed1999

A numerical study of blood flow patterns in anatomically realistic and simplified end-to-side anastomoses.

PURPOSE: Recently, some numerical and experimental studies of blood flow in large arteries have attempted to accurately replicate in vivo arterial geometries, while others have utilized simplified models. The objective of this study was to determine how much an anatomically realistic geometry can be simplified without the loss of significant hemodynamic information. METHOD: A human femoral-popliteal bypass graft was used to reconstruct an anatomically faithful finite element model of an end-to-side anastomosis. Nonideal geometric features of the model were removed in sequential steps to produce a series of successively simplified models. Blood flow patterns were numerically computed for each geometry, and the flow and wall shear stress fields were analyzed to determine the significance of each level of geometric simplification. RESULTS: The removal of small local surface features and out-of-plane curvature did not significantly change the flow and wall shear stress distributions in the end-to-side anastomosis. Local changes in arterial caliber played a more significant role, depending upon the location and extent of the change. The graft-to-host artery diameter ratio was found to be a strong determinant of wall shear stress patterns in regions that are typically associated with disease processes. CONCLUSIONS: For the specific case of an end-to-side anastomosis, simplified models provide sufficient information for comparing hemodynamics with qualitative or averaged disease locations, provided the "primary" geometric features are well replicated. The ratio of the graft-to-host artery diameter was shown to be the most important geometric feature. "Secondary" geometric features such as local arterial caliber changes, out-of-plane curvature, and small-scale surface topology are less important determinants of the wall shear stress patterns. However, if patient-specific disease information is available for the same arterial geometry, accurate replication of both primary and secondary geometric features is likely required.

Anastomosis, Surgical↗
PubMed1999

Complementation of conjugation functions of Streptomyces lividans plasmid pIJ101 by the related Streptomyces plasmid pSB24.2.

A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24. 2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.

Amino Acid Sequence↗
PubMed1999

Growth kinetics of genetically engineered E. coli DH 5 cells in artificial cell APA membrane microcapsules: preliminary report.

This paper describes the growth kinetics of genetically engineered E. coli DH5 cells inside the APA membrane artificial cells. The APA microcapsule membrane found does not significantly affects the growth of the encapsulated E. coli DH5 cells. The total protein production of the E. coli DH5 cells inside the APA microcapsules were not significantly different from that of the bacterial cells grown in the free bacterial media. The result also show that the log phase APA artificial cells containing genetically engineered E. coli DH5 would be highly effective for the conversion of various external metabolites.

Alginates↗
PubMed1998

Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA.

Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes.

Cell-Free System↗
PubMed1998

Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites.

Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1 (REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Delta apn2Delta strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase zeta mediates the mutagenic bypass of AP sites.

Amino Acid Sequence↗
PubMed1998

ATP-dependent assembly of a ternary complex consisting of a DNA mismatch and the yeast MSH2-MSH6 and MLH1-PMS1 protein complexes.

MSH2 and MSH6 proteins exist as a stable complex, as do the MLH1 and PMS1 proteins. To study the mismatch binding properties of the MSH2-MSH6 complex and to examine its functional interaction with the MLH1-PMS1 complex, these protein complexes were purified to near homogeneity from overproducing yeast strains. As has been reported previously, the purified MSH2-MSH6 complex binds DNA substrates containing a G/T mismatch and insertion/deletion mismatches, but the binding affinity for the latter decreases as the size of the extrahelical loop increases. Addition of ATP or the nonhydrolyzable ATPgammaS reduces binding of the MSH2-MSH6 complex to the DNA substrates markedly. Here, we show that MSH2-MSH6 forms a ternary complex with MLH1-PMS1 on a mismatch containing DNA substrate. The formation of this ternary complex requires ATP, which can be substituted by ATPgammaS, suggesting that ATP binding alone is sufficient for ternary complex formation. Thus, it appears that ATP binding by the MSH2-MSH6 complex induces a conformation that is conducive for the interaction with MLH1-PMS1 complex, leading to the formation of the ternary complex.

Adenosine Triphosphate↗
PubMed1998

Crystal structure of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Rad6 at 2.6 A resolution.

The Saccharomyces cerevisiae ubiquitin-conjugating enzyme (UBC) Rad6 is required for several functions, including the repair of UV damaged DNA, damage-induced mutagenesis, sporulation, and the degradation of cellular proteins that possess destabilizing N-terminal residues. Rad6 mediates its role in N-end rule-dependent protein degradation via interaction with the ubiquitin-protein ligase Ubr1 and in DNA repair via interactions with the DNA binding protein Rad18. We report here the crystal structure of Rad6 refined at 2.6 A resolution to an R factor of 21.3%. The protein adopts an alpha/beta fold that is very similar to other UBC structures. An apparent difference at the functionally important first helix, however, has prompted a reassessment of previously reported structures. The active site cysteine lies in a cleft formed by a coil region that includes the 310 helix and a loop that is in different conformations for the three molecules in the asymmetric unit. Residues important for Rad6 interaction with Ubr1 and Rad18 are on the opposite side of the structure from the active site, indicating that this part of the UBC surface participates in protein-protein interactions that define Rad6 substrate specificity.

Amino Acid Sequence↗
PubMed1998

The DNA-dependent ATPase activity of yeast nucleotide excision repair factor 4 and its role in DNA damage recognition.

Saccharomyces cerevisiae RAD7 and RAD16 genes function together in the nucleotide excision repair of transcriptionally inactive DNA. The RAD7- and RAD16-encoded proteins exist as a tight complex named nucleotide excision repair factor 4 or NEF4. Previously, we showed that NEF4 binds UV-damaged DNA with high specificity and with a dependence upon ATP and that inclusion of NEF4 to the reconstituted nucleotide excision repair system consisting of purified NEF1, NEF2, NEF3, and replication protein A results in marked stimulation of damage-specific DNA incision. Here we show that NEF4 possesses an ATPase activity that is entirely dependent on a DNA cofactor and that double-stranded DNA is twice as effective as single-stranded DNA in activating ATP hydrolysis. Even though DNA binding is promoted by the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate) (ATPgammaS), damage binding is more proficient with ATP than with ATPgammaS. Interestingly, UV irradiation of double-stranded DNA results in a pronounced attenuation of the ATPase activity. Taken together, our results suggest a model in which ATP hydrolysis by NEF4 fuels the translocation of NEF4 on DNA in search of UV lesions and damage binding by NEF4 leads to a down-regulation of the ATPase activity. Damage-bound NEF4 could then serve as a nucleation point for the assembly of other repair components.

Adenosine Triphosphatases↗
PubMed1998

Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication.

The RTH1(RAD27) gene of Saccharomyces cerevisiae encodes a structure-specific endonuclease that cleaves 5'-ended single-stranded DNA at its junction with duplex DNA. Genetic and biochemical studies have indicated a role of Rth1 nuclease in the removal of RNA primers formed during DNA replication. The rth1Delta mutation confers temperature-sensitive lethality, and increases sensitivity to alkylating agents. The instability of repetitive DNA is greatly enhanced in the rth1Delta mutant. The conditional lethality of the rth1Delta mutation indicates that another nuclease can function in DNA replication in the absence of RTH1. RAD2, a homolog of RTH1, is required for nucleotide-excision repair. Here, we examine three other homologs of RTH1/RAD2 - YEN1, EXO1, and DIN7. Deletion of any of these genes in the rth1Delta strain has no effect on cell viability, suggesting the involvement of another, and as yet unidentified, nuclease in the maturation of Okazaki fragments. Our data also indicate that only RTH1 functions in the repair of alkylation damage. Deletions of YEN1, EXO1, DIN7, or RAD2, either singly or when combined with one another and with the rth1Delta mutation, have no effect on the rate of instability of dinucleotide repeats or on the rate of formation of large duplications in the CAN1 gene. These data provide evidence of a high degree of specificity for the role of RTH1 in DNA replication and in base-excision repair, and for the requirement of RAD2 in nucleotide-excision repair. The possibility that both Rth1 and Exo1 function in DNA mismatch repair is discussed.

Alkylation↗