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Biomedical subjects

A Srinivasan

Publications and source records attributed to A Srinivasan.

At least 163 records · Page 9Linked to original sources
PubMed1997

The discovery of indicator variables for QSAR using inductive logic programming.

A central problem in forming accurate regression equations in QSAR studies is the selection of appropriate descriptors for the compounds under study. We describe a novel procedure for using inductive logic programming (ILP) to discover new indicator variables (attributes) for QSAR problems, and show that these improve the accuracy of the derived regression equations. ILP techniques have previously been shown to work well on drug design problems where there is a large structural component or where clear comprehensible rules are required. However, ILP techniques have had the disadvantage of only being able to make qualitative predictions (e.g. active, inactive) and not to predict real numbers (regression). We unify ILP and linear regression techniques to give a QSAR method that has the strength of ILP at describing steric structure, with the familiarity and power of linear regression. We evaluated the utility of this new QSAR technique by examining the prediction of biological activity with and without the addition of new structural indicator variables formed by ILP. In three out of five datasets examined the addition of ILP variables produced statistically better results (P < 0.01) over the original description. The new ILP variables did not increase the overall complexity of the derived QSAR equations and added insight into possible mechanisms of action. We conclude that ILP can aid in the process of drug design.

Animals↗
PubMed1997

Relative mutagenicities of gaseous nitrogen oxides in the supF gene of pSP189.

Gaseous nitric oxide (NO), an environmental pollutant found in cigarette smoke and diesel exhaust, has been shown to generate mutations in aerobic in vitro assays. The objective of this study was to identify which oxides of nitrogen, formed in the gaseous phase from NO, possess mutagenic activity. Samples of the plasmid pSP189, in 1 M HEPES buffer, pH 7.4, were exposed to preparations of nitrogen dioxide (NO2), dinitrogen trioxide (N2O3) or an air control. The gas mixtures were formed in a gas-tight syringe and were then introduced into 1 l flasks. The plasmid solution was introduced immediately afterwards. Transformation of Escherichia coli strain MBM7070 with the treated plasmids allowed analysis of mutation frequencies and the types of mutations induced in the target supF gene. The mutation frequency resulting from NO2 exposure was not different from that of the control. However, N2O3 produced a substantial number of mutations. The mutation frequency and the types of mutations were found to depend on the length of time that the gases were allowed to incubate in the syringe prior to introduction into the 1 l flasks (mutation frequency was maximal at approximately 2 min). The most prevalent mutations were AT-->GC transitions (68%), followed by GC-->AT transitions (30%), similar to previous findings when pure NO was bubbled through pSP189 solutions. These results suggest that it is N2O3, rather than NO2, that is the most likely source of mutagenic potential from gaseous nitrogen oxides.

Base Sequence↗
PubMed1997

Simian virus 40 T antigen can regulate p53-mediated transcription independent of binding p53.

A simian virus 40 (SV40) T-antigen mutant containing only the N-terminal 136 amino acids, able to bind to Rb and p300 but not p53, partially inhibited p53-mediated transcription without affecting the ability of p53 to bind DNA. These results suggest that SV40 T antigen can regulate p53-mediated transcription either directly through protein-protein association or indirectly through interaction with factors which may function to confer p53-mediated transcription.

Animals↗
PubMed1997

The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain.

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.

Adenosine Triphosphatases↗
PubMed1996

Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis.

The ICE/CED-3 family of proteases has been implicated in playing a fundamental role in programmed cell death. Bcl-2 protein represses a number of apoptotic death programs, but the biochemical mechanism of its action is not known. We investigated the activation of ICE/CED-3 proteases induced by three apoptotic stimuli (staurosporine, ceramide, and serum withdrawal) in the neuronal cell line GT1-7 and in cells overexpressing Bcl-2. Rapid activation of a 17 kDa subunit of an activated member of the ICE/CED-3 family is demonstrated by affinity-labeling GT1-7 extracts from apoptotic controls cells with a biotinylated ICE/CED-3 inhibitor. This activation corresponds to an increased ICE/CED-3-like protease activity in extracts measured by a fluorogenic substrate assay. In a cell-free system, these extracts induce apoptotic morphological changes in intact nuclei. All three activities are readily inhibited by treatment of control extracts with ICE/CED-3-like protease inhibitors. Overexpressed Bcl-2 inhibits the activation of the 17 kDa protein, the ICE/CED-3-like protease activity in the fluorogenic assay, and the induction of apoptotic morphological changes in HeLa nuclei in the cell-free system, similar to results obtained with ICE/CED-3 protease inhibitors. At the mRNA level, overexpression of Bcl-2 did not alter expression of five members of the ICE/CED-3 family: CPP32, ICE, Mch 2, Nedd 2, and TX. Overexpression of Bcl-2 prevented the apoptosis-induced processing of pro-Nedd 2 to the cleaved form. These data suggest that Bcl-2 participates upstream from the function of ICE/CED-3 proteases and may inhibit apoptosis by preventing the post-translational activation of ICE/CED-3 proteases.

Animals↗
PubMed1996

The RNA subunit of ribonuclease P from the zebrafish, Danio rerio.

A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.

Animals↗
PubMed1996

Structure-activity relationships derived by machine learning: the use of atoms and their bond connectivities to predict mutagenicity by inductive logic programming.

We present a general approach to forming structure-activity relationships (SARs). This approach is based on representing chemical structure by atoms and their bond connectivities in combination with the inductive logic programming (ILP) algorithm PROGOL. Existing SAR methods describe chemical structure by using attributes which are general properties of an object. It is not possible to map chemical structure directly to attribute-based descriptions, as such descriptions have no internal organization. A more natural and general way to describe chemical structure is to use a relational description, where the internal construction of the description maps that of the object described. Our atom and bond connectivities representation is a relational description. ILP algorithms can form SARs with relational descriptions. We have tested the relational approach by investigating the SARs of 230 aromatic and heteroaromatic nitro compounds. These compounds had been split previously into two subsets, 188 compounds that were amenable to regression and 42 that were not. For the 188 compounds, a SAR was found that was as accurate as the best statistical or neural network-generated SARs. The PROGOL SAR has the advantages that it did not need the use of any indicator variables handcrafted by an expert, and the generated rules were easily comprehensible. For the 42 compounds, PROGOL formed a SAR that was significantly (P < 0.025) more accurate than linear regression, quadratic regression, and back-propagation. This SAR is based on an automatically generated structural alert for mutagenicity.

Algorithms↗
PubMed1996

Androgen regulation of ribosomal RNA synthesis in LNCaP cells and rat prostate.

Androgen-dependent growth of prostate tissue has been well documented. An additional prerequisite for cellular growth is the accumulation of ribosomes. It is thus reasonable to hypothesize that ribosomal DNA (rDNA) transcription in prostate tissue must be stimulated by androgen either directly or indirectly. This hypothesis was tested using both LNCaP cells, an androgen-dependent tissue culture line and in a rat animal model. Nuclear run-on assays confirmed that the administration of DHT to LNCaP cells resulted in a two- to three-fold increase in the rate of rRNA synthesis when compared to cells maintained in the absence of androgen. Enzymatic analysis and Western blots were carried out to measure the amount (activity and mass) of RNA polymerase I in DHT treated LNCaP cells. These assays demonstrated that neither the catalytic activity of RNA polymerase I nor the amount of the enzyme varied in response to DHT. However, Western blots revealed that the amount of the auxiliary RNA polymerase I transcription factor UBF, was significantly increased (two- to three-fold) in cells grown in the presence of DHT. Similar experiments were carried out with prostatic tissue obtained from orchiectomized rats maintained on either placebo or testosterone pellets. In this model, both the catalytic activity as well as the amount of RNA polymerase I protein decreased. However, in agreement with the tissue culture model, UBF protein decreased in prostates from orchiectomized rats and was maintained in animals supplemented with testosterone. These lines of evidence are consistent with the hypothesis that androgens stimulate rRNA synthesis by increasing the quantities of the components of the rDNA transcription system.

Animals↗
PubMed1996

Expression of recombinant alpha Ains-crystallin and not alpha A-crystallin inhibits bacterial growth.

alpha A-Crystallin and alpha Ains-crystallin are derived from the alpha A-crystallin gene via alternative splicing. They are identical except for the presence of a polypeptide, 23 amino acids long, encoded by the 'insert' exon. Evolutionary logic would suggest that the insertion of a 23 amino acid peptide in the middle of alpha A-crystallin, a protein evolving more slowly than either histone H1, cytochrome c or hemoglobin, would lead to appreciable structural and functional changes. However, based on physico-chemical studies, it is presently believed that alpha A-crystallin and alpha Ains-crystallin are functionally equivalent and that the presence of the 'insert' peptide in alpha Ains-crystallin is inconsequential. We report here that the independent expression of recombinant alpha Ains-crystallin, and not alpha A-crystallin, inhibits growth of the bacterial host. These observations were confirmed in coexpression experiments, wherein both the proteins were expressed in the same cell. Interestingly, growth inhibition is reversible. Importantly, the data demonstrate that it is catalytic amounts and not the gross accumulation of alpha Ains-crystallin which causes growth inhibition. Given the prior knowledge that alpha A-crystallin and alpha Ains-crystallin differ by a peptide of 23 amino acids, these data suggest that the "insert peptide' in alpha Ains-crystallin imparts properties on this protein that are different from alpha A-crystallin.

Animals↗
PubMed1996

Aetiology of pneumonia in hospitalized children.

One-hundred-and-thirty-two children with clinical and radiological evidence of bronchopneumonia/pneumonia were studied over a 1-year period for isolation/detection of bacterial and viral aetiological pathogens. Throat swab, nasopharyngeal aspirate (NPA), and lung aspirate were studied for bacterial and viral cultures. NPA was also subjected to latex agglutination test (LA) for H. influenzae and S. pneumoniae; and immunofluorescent technique (IFAT) and enzyme immunoassay (EIA) for respiratory syncytial virus (RSV). Blood culture for bacterial pathogens, and LA of blood and urine was also undertaken. Haemophilus influenzae was the commonest organism (15 per cent) isolated as the sole pathogen followed by RSV (14 per cent), Klebsiella (13 per cent) and S. pneumoniae (12 per cent). E. coli was the commonest organism (50 per cent) in infants <3 months and was closely followed by RSV (44 per cent), Klebsiella (25 per cent), and S. pneumoniae (18 per cent). Isolation rate of E. coli gradually declined with age. RSV (47 per cent) and H. influenzae (31 per cent) were the commonest organisms between 7 and 24 months. S. pneumoniae and Staph. aureus were common bacterial pathogens identified in all age groups with maximum isolation of 20 and 40 per cent, respectively, in children more than 5 years. Isolation of E. coli, Klebsiella and Staph. aureus was highest from NPA culture, while as S. pneumoniae and H. influenzae were most often detected by LA. Out of 12 cases from whom a lung aspirate was collected, bacterial pathogen could be isolated in six cases (50 per cent). Detection of RSV by EIA was higher than by culture or IFAT. Most of the organisms were resistant to chloramphenicol except for H. influenza. All the isolates of S. pneumoniae were sensitive to all the antibiotics. Bacterial pathogens were isolated/detected in 74 per cent of cases and RSV was the aetiological agent in 49 per cent of cases investigated for viral aetiology. Higher detection rate of RSV is attributed to selection of cases in winter months during a period of suspected epidemic of RSV.

Bacterial Infections↗
PubMed1996

Prediction of rodent carcinogenicity bioassays from molecular structure using inductive logic programming.

The machine learning program Progol was applied to the problem of forming the structure-activity relationship (SAR) for a set of compounds tested for carcinogenicity in rodent bioassays by the U.S. National Toxicology Program (NTP). Progol is the first inductive logic programming (ILP) algorithm to use a fully relational method for describing chemical structure in SARs, based on using atoms and their bond connectivities. Progol is well suited to forming SARs for carcinogenicity as it is designed to produce easily understandable rules (structural alerts) for sets of noncongeneric compounds. The Progol SAR method was tested by prediction of a set of compounds that have been widely predicted by other SAR methods (the compounds used in the NTP's first round of carcinogenesis predictions). For these compounds no method (human or machine) was significantly more accurate than Progol. Progol was the most accurate method that did not use data from biological tests on rodents (however, the difference in accuracy is not significant). The Progol predictions were based solely on chemical structure and the results of tests for Salmonella mutagenicity. Using the full NTP database, the prediction accuracy of Progol was estimated to be 63% (+/- 3%) using 5-fold cross validation. A set of structural alerts for carcinogenesis was automatically generated and the chemical rationale for them investigated- these structural alerts are statistically independent of the Salmonella mutagenicity. Carcinogenicity is predicted for the compounds used in the NTP's second round of carcinogenesis predictions. The results for prediction of carcinogenesis, taken together with the previous successful applications of predicting mutagenicity in nitroaromatic compounds, and inhibition of angiogenesis by suramin analogues, show that Progol has a role to play in understanding the SARs of cancer-related compounds.

Animals↗
PubMed1996

Proto-oncogene expression in cAMP and TPA-mediated neuronal differentiation in a human retinal cell line KGLDMSM.

PURPOSE: A human retinal cell line, KGLDMSM, developed by SV-40T antigen gene transfection, is stable in culture for a long period, unlike the primary cells. The cell line shows some degree of morphological differentiation with limited extension of stublike neurites upon transfer to defined medium. In our effort to explore genes implicated in neuritic extension and neuronal differentiation seen in response to cAMP and TPA, we have analyzed time dependent induction of a variety of proto-oncogenes: c-myc, H-ras, c-ras, and c-fos. METHODS: Cells were adapted to grow in defined media and exposed to differentiation inducing agents cAMP, TPA, Retinoic Acid, and sodium butyrata. Cells were assessed for phenotypic changes and altered expression of proto-oncogenes as evaluated by Northern Blot analysis and immunocytochemistry. RESULTS: Exposure of the cells to cAMP and TPA induced dramatic changes, with 100% of the cells extending neuritic processes. However, other differentiation inducing agents such as retinoic acid and sodium butyrata failed to elicit any response. We report that agents that promote neuritic extension also induce expression of c-fos. Transcriptional activation of c-fos in response to cAMP (30 min) and TPA (1hr) is also accompanied by expression of fos gene product as evaluated by using fos antibody. No fos expression was seen in uninduced cells. CONCLUSION: In retinal cell line KGLDMSM, agents that enhance neuronal differentiation (cAMP, TPA) also induce c-fos expression. Expression of c-fos may be a necessary prerequisite in neuronal differentiation and the established retinal cell line offers an excellent cell model for dissecting the molecular events underlying neuronal differentiation.

Cell Differentiation↗
PubMed1995

The carboxy-terminal domain is essential for stability and not for virion incorporation of HIV-1 Vpr into virus particles.

Vpr is one of the auxiliary gene products encoded by HIV-1 genome. Vpr is a 14-kDa protein and exhibits several interesting characteristics including incorporation into virus particles, oligomerization, localization in the nucleus, and positive regulation of virus replication in primary cells. In an effort to define the structure-function relationship of Vpr, the role of the C-terminus of Vpr was investigated. Site-specific mutagenesis involving deletion, insertion, and substitution of residues at the C-terminus was utilized to generate variants of Vpr. Mutations introduced at the C-terminus affected properties of Vpr in different ways: (i) Vpr containing amino acids 1-72 showed the virion incorporation phenotype, indicating that the C-terminus is not essential for this function, (ii) the C-terminus contributes to the stability of Vpr, and (iii) substitution mutagenesis involving the basic residues showed stability similar to that of wild type, indicating the lack of involvement of these residues in this biochemical property of Vpr. The data generated in this study and our early mutagenic analyses on Vpr suggest that domains noncontiguous in primary sequence contribute to the stability of Vpr through overall conformation of the protein.

Amino Acid Sequence↗
PubMed1995

Functional analysis of HIV-1 Vpr: identification of determinants essential for subcellular localization.

Vpr is a conserved HIV-1 auxiliary protein that localizes to the nuclear region of cells. Vpr is also present in virions, and it is directed into the assembling virus when coexpressed with Gag. Each of these two localization activities may be important for Vpr function, and we recently identified regions of Vpr that are critical for virion incorporation. In this study we analyzed the Vpr domains involved in subcellular localization. Immunofluorescence staining of transfected cells showed that wild-type Vpr localized exclusively to the nuclear region. Mutations in the N-terminal domain that were designed to disrupt a predicted alpha-helical structure resulted in aberrant localization, while conservative substitutions showed a wild-type pattern. A region in the central portion of the protein also has the potential for helical structure, and mutagenesis of two conserved amino acids in this domain (A59, H71) impaired localization, while substitution of a third (Q65) did not. In contrast, neither the conserved Gly and Cys at positions 75-76 nor the C-terminal basic residues (R87, K95) were necessary for nuclear localization. In addition, two-residue insertions within and between the two putative helices disrupted localization but insertion in the C-terminal region did not. Thus, Vpr's subcellular localization function depends on the two putative helical domains but is independent of the conserved Gly-Cys motif and of specific C-terminal basic residues.

Amino Acid Sequence↗
PubMed1995

Role of the conserved dipeptide Gly75 and Cys76 on HIV-1 Vpr function.

Vpr is one of the accessory proteins encoded by the HIV-1 genome. Several interesting features associated with Vpr include incorporation into virus particles, ability to oligomerize, localization in the nucleus, and positive effect on virus production and replication. In order to understand the structure-function relationship of Vpr, we have analyzed the role of the Gly75 and Cys76 (GC) residues which are highly conserved in HIV-1 Vpr and in Vpr and Vpx of HIV-2/SIV. We have generated several substitution mutants involving this dipeptide and have evaluated for expression, stability, nuclear localization, and virion incorporation of Vpr. Our data demonstrate that the GC residues are not essential for virion incorporation and nuclear localization of Vpr. Serine substitution for Cys, however, restricted the localization of Vpr in the cytoplasm without affecting the Gag-directed incorporation of Vpr into virus-like particles. Interestingly, the cysteine-substituted mutants showed altered stability in comparison to the wild type, and substitution mutants for glycine showed minimal effect on stability. These results indicate that the glycine and cysteine do not play a role in nuclear localization or virion incorporation properties of Vpr and further suggest that these two functions of Vpr may not be interdependent.

Amino Acid Sequence↗
PubMed1995

HIV infection decreases intracellular nicotinamide adenine dinucleotide [NAD].

We report that HIV-1 infection of human cells in vitro leads to significant decreases in the intracellular concentration of NAD. This decrease varies with viral load and HIV strain. In tissue culture, cells lacking CD4 receptors or cells incubated with heat inactivated virus do not demonstrate this decrease in NAD. Nicotinamide, the amide form of the vitamin niacin, increases intracellular NAD levels in uninfected cells as expected. Our data demonstrate that nicotinamide also maintains increased intracellular NAD concentrations in HIV infected cells. We conclude that HIV induces a state of intracellular pellagra which is reversed by the administration of nicotinamide.

Cell Line↗
PubMed1995

Nicotinamide inhibits HIV-1 in both acute and chronic in vitro infection.

HIV-1 infected patients can manifest a number of poorly understood conditions including dermatitis, dementia, and diarrhea. These conditions are in some ways suggestive of pellagra, the syndrome associated with niacin depletion. We demonstrate here that nicotinamide, the amide form of niacin, inhibits HIV-1 infection in cell culture. Neither nicotinic acid which is the alternative form of niacin, nor thiamine (another B complex vitamin), shows a similar degree of inhibition in tissue culture. This inhibition occurs in both primary cells and in established cell lines. In vitro models of acute and chronic HIV infection are demonstrated here to be inhibited by nicotinamide in a dose dependent manner when added in millimolar concentrations.

Antiviral Agents↗