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N Kaushik

Publications and source records attributed to N Kaushik.

11 recordsLinked to original sources
PubMed1995

Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity.

In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.

Arginine↗
PubMed1995

Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase.

In order to define the role of Gln151 in the polymerase function of HIV-1 RT, we carried out site-directed mutagenesis of this residue by substituting it with a conserved (Q151N) and a nonconserved residue (Q151A). Q151N exhibited properties analogous to those of the wild-type enzyme, while Q151A has severely impaired polymerase activity. The Q151A mutant exhibited a 15-100-fold reduction in kcat with RNA [poly(rC) and poly(rA)] templates, while only a 5-fold reduction could be seen with the DNA [poly(dC)] template. Most interestingly, the affinity of the Q151A mutant for dNTP substrate remained unchanged with RNA templates, but a significant increase in Km was noted with the DNA template. The binding affinity of Q151A for DNA remained unchanged, as judged by photoaffinity cross-linking. However, unlike the wild-type enzyme, the Q151A mutant failed to catalyze the nucleotidyl transferase reaction onto the primer terminus of the covalently immobilized template-primer. The enzyme showed profoundly altered divalent cation preference from Mg2+ to Mn2+. These results strongly implicate Q151 of HIV-1 RT in the substrate dNTP binding function and possibly in the following chemical (catalytic) step. The effects of the mutation seem to be through Q151 of the p66 catalytic subunit, as p66WTt/p51Q151A retains the wild-type kinetic constants and nucleotidyl transferase activity. In contrast, p66Q151A/p51WT is indistinguishable from Q151A (mutated in both subunits). A model of the ternary complex (enzyme-template-primer and dNTP) has been used to infer the possible mode by which Q151 may interact with the base moiety of the substrate as well as with Arg72, a residue present within the active site of HIV-1 RT.

Amino Acid Sequence↗
PubMed1995

Genetic map of the region surrounding the retrovirus restriction locus, Fv1, on mouse chromosome 4.

The Friend virus susceptibility-1 (Fv1) gene maps to mouse Chromosome (Chr) 4 close to a cluster of four endogenous murine leukemia viruses (MLVs). To investigate the feasibility of cloning Fv1 by a positional approach, we have performed an extensive genetic analysis of this region of Chr 4. We have typed 368 backcross mice for the four proviruses, Nppa, Lck, and D4Smh6b. Recombinant animals were screened in a hierarchical fashion with a variety of other markers, including Fv1 and the isozyme marker Gpd1. A detailed genetic map of the region surrounding Fv1 was derived. Three markers, Xmv9, Nppa, and Iap3rc11, were identified that showed no recombination with Fv1. By combining backcross and recombinant inbred strain data, we estimated that Xmv9 and Nppa must lie within 0.6 cM of one another and Fv1.

Animals↗
PubMed1992

Supportive role of image analysis and DNA ploidy pattern in the diagnosis of thyroid tumors.

To evaluate the supportive role of image cytometry and DNA ploidy pattern in the diagnosis of thyroid tumors, a preliminary study was performed on fine needle aspirates of 30 cases. Of these, 10 cases each were of colloid goiter, follicular neoplasm, and papillary carcinoma. The nuclear area and DNA value of 50 cells in each case were measured. The mean nuclear area in colloid goiter (69.50 + 12.62 sq.microns) was significantly lower than the mean nuclear area in a follicular neoplasm (88.71 + 15.51 sq.microns) (P less than 0.05). Similar differences between the mean nuclear area in colloid goiter and papillary carcinoma (124.0 + 12.27 sq microns) was also highly significant (P less than 0.001). The results obtained by image cytometry were compared with estimated DNA ploidy pattern of follicular cells from the same cases. All colloid goiter had mean nuclear area below 100 sq.microns with diploid DNA value. However, papillary carcinoma showed aneuploid DNA pattern in eight cases (80.0%), but mean nuclear area was above 100 sq.microns. A diagnostically useful finding obtained in two of the 10 cases of follicular neoplasm was the association of aneuploid DNA pattern with mean nuclear area of the follicular cells above 100 sq.microns indicating a high probability of carcinoma and thus demanding an urgent open biopsy. These cases were readily distinguished from other cases of the same category showing diploid DNA pattern and mean nuclear area of follicular cells below 100 sq.microns.

Cell Nucleus↗
PubMed1991

Quantitative analysis of nuclear area variation in benign and malignant breast fine needle aspirates.

The measurement of nuclear area was carried out in 30 benign and 32 malignant breast lumps using Omnicon Alpha 500 Image Analyzer. The mean nuclear area of duct cells in malignant group was greater (157.6 +/- 58.64 sq.microns with a peak around 140 sq.microns) and more heterogenous within and amongst cases than observed in duct cells from most of the cases of fibroadenoma (85.05 ae 14.2 sq.microns with a peak around 80 sq.microns). Taking into consideration 110 sq.microns as a differentiating limit, a significant difference was observed between benign and malignant conditions (p). Similarly taking 118 sq.microns as differentiating limit duct cell carcinomas could be divided into two groups i.e. 9(28.1%) cases of small nuclear type with a range of 80-118 sq.microns and 23(71.9%) cases of large nuclear type with a range of 118-320 sq microns .6(18.8%) cases with small nuclei had an overlap with fibroadenoma. Although 13(72.2%) cases of large nuclear type carcinomas had lymph node metastasis as against 4(44.4%) in small nuclear group, the difference was not statistically significant.

Adenofibroma↗
PubMed1990

Cervical intraepithelial glandular neoplasia diagnosis and significance in relation to squamous precancerous lesions.

The endocervical epithelium of histologically confirmed dysplasia and carcinoma-in-situ cases of cervix uteri was examined for the presence or absence of intraepethelial glandular Neoplasia (CIGN). Results from 80 cases were compared with equal number of controls who were being operated for reasons other than Neoplasia. In the present communication we have used the criteria of Nuclear enlargement and elongation, hyperchromasia, stratification, increased N/C ratio, pleomorphism and dence clumping to distinguish CIGN from normal endocervical epithelial lining using the criteria mentioned above we recorded CIGN in eleven cases of test group while in two of controls which implied a statistically significant (p less than .5) association of CIGN with case series as compared to controls. Among the test group; 1, 2 (15.3%), 3 (23%) and 5 (10%) cases were associated with mild, moderate, sever dysplasia and carcinoma-in-situ respectively.

Adult↗
PubMed1986

Trypsin-sensitive plasmodia in the liver of post-infection rats and rhesus monkeys.

Kupffer cells from the liver and erythrocytes from peripheral blood were collected at the post-patent period from albino rats infected earlier with Plasmodium berghei and rhesus monkeys infected earlier with P. cynomolgi var. bastianelli or P. knowlesi. The cells were subinoculated into individual normal recipients. These recipients subsequently showed parasitaemia in their circulation. The parasites present in Kupffer cell preparations were found to be sensitive to trypsin treatment, while those in erythrocytes were found to be resistant to trypsin treatment. This differential sensitivity of parasites to trypsin was observed in all the three species of plasmodia studied so far.

Animals↗