Search PubMed⌕ Search

Biomedical subjects

N Kaushik

Publications and source records attributed to N Kaushik.

52 records · Page 3Linked to original sources
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↗
PubMed1994

Photoaffinity labeling of DNA template-primer binding site in Escherichia coli DNA polymerase I. Identification of involved amino acids.

We have used two self-annealing template-primers (TPs) to covalently cross-link the Klenow fragment of Escherichia coli DNA polymerase I in its polymerase mode. The specificity of cross-linking is demonstrated by the observation that other template-primers, but not the template or primer alone, readily compete with self-annealing TPs. The enzyme-TP covalent complex is catalytically active and can incorporate one nucleotide on the primer terminus of the immobilized template-primer. Using a peptide mapping approach, we have identified a 17-amino acid tryptic peptide spanning residues 759-775 as a major constituent of the TP binding domain. Amino acid sequence analysis further revealed that Ile-765, Tyr-766 in the O-helix and Ser-769, Phe-771 in the O1-helix of the three-dimensional crystal structure of the Klenow fragment constitute the attachment site for TP.

Affinity Labels↗
PubMed1994

Role of lysine 758 of Escherichia coli DNA polymerase I as assessed by site-directed mutagenesis.

Lys-758 of Escherichia coli DNA polymerase I has been implicated in the process of substrate dNTP binding (Basu, A., and Modak, M. J. (1987) Biochemistry 26, 1704-1709). To confirm and define the role of Lys-758 in the catalytic mechanism, we carried out site-directed mutagenesis of this residue. Catalytic activity of the purified mutant enzymes, K758A and K758R, showed severe reduction in the polymerase activity but little difference in the 3'-->5' exonuclease activity. Most interestingly, the catalytic ability of both mutant enzymes was maximally affected (300-1,000-fold decrease in kcat) with poly(dA).(dT)15 as template-primer (TP), whereas the ability to use poly(dC) templates decreased by only 20-fold in K758A and remained nearly unchanged with K758R. Kinetic characterization showed that Km(dNTP) increased moderately only with K758A, whereas Kd(TP) remained unchanged for both the mutants. However, binary complex formation between K758A and dNTP, but not between K758A and TP, was severely reduced. Analysis of the processive mode of DNA synthesis by K758A indicated that the mutant enzyme pauses at dA bases but does not dissociate from TP, suggesting a defect in its translocation ability. Thus, Lys-758 in polymerase I appears to participate in two distinct functions: (a) it facilitates the dNTP binding, and (b) it is required for the translocation along the template polynucleotide.

Base Sequence↗
PubMed1994

Intracisternal A-type particle elements as genetic markers: detection by repeat element viral element amplified locus-PCR.

We describe a novel, PCR-based technique termed REVEAL-PCR for examining the inheritance of intracisternal A-type particles (IAP). Amplifications use an unlabeled primer to SINE repeats and a radiolabeled primer to the IAP long terminal repeat; labeled products, which can be resolved on sequencing gels, are formed when IAPs lie in proximity to SINEs. With this technique we have identified a total of 124 polymorphisms in the BXH and CXS recombinant inbred strains. We suggest that this method will be equally applicable for examining other gene families present at around a thousand copies per genome.

Animals↗
PubMed1993

Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli. The significance of Arg682 in catalysis.

We have reported that a domain containing Arg682 in the Klenow fragment of Escherichia coli DNA polymerase I (pol I) is important for the template-dependent dNTP-binding function [Pandey, V.N., Kaushik, N. A., Pradhan, D. S. & Modak, M. J. (1990) J. Biol. Chem. 265, 3679-3884]. In order to further define the role of Arg682 in the catalytic process, we have performed site-directed mutagenesis of this residue. For this purpose the Klenow-coding region of the DNA-pol-I gene was selectively amplified from the genomic DNA of E. coli and was cloned in an expression vector, pET-3a. This clone under appropriate conditions overproduces the Klenow fragment in E. coli. Using this clone (pET-3a-K) as the template, two mutant polymerase clones were constructed in which arginine has been replaced with either alanine, [R682A] pol I, or lysine [R682K] pol I. Both mutant enzymes showed significantly lower specific activity as compared to the wild-type enzyme. The kinetic analyses of the mutant enzymes indicated a 3-4-fold increase in the Km for the substrate dNTP, a 20-25-fold decrease in the Vmax and an overall decrease in the processive nature of DNA synthesis in both the mutant enzymes. The reverse mutation of Ala682 to the wild-type form Arg682 fully restored the processive nature and the polymerase activity of the enzyme. These observations suggest that the positively charged guanidino group in the side chain of Arg682 is catalytically important but not absolutely essential for synthesis of DNA. Furthermore it appears to maintain high processivity of the DNA synthesis catalyzed by the enzyme.

Arginine↗
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↗
PubMedDate not supplied

An isolated left common carotid artery from the main pulmonary artery: possible malseptation of the truncoaortic sac.

An isolated left common carotid artery (LCA) is an extremely rare condition with only four reported cases. In each case, the isolated carotid artery connects to the right or left pulmonary artery via the ductus arteriosus and the embryologic basis for the abnormalities is believed to reflect an error in the development of the branchial arches. We present a case of an isolated LCA connecting to the main pulmonary artery in association with a right aortic arch and an anomalous origin of the left subclavian artery from the descending aorta. The left ligamentus arteriosus was identified separately. This may represent a disturbance in the septation of the truncoaortic sac secondary to abnormal migration of neural crest cells rather than a pure developmental anomaly of the branchial arches.

Aorta, Thoracic↗
PubMedDate not supplied

A survey of trinucleotide/tandem repeat-containing transcripts (TNRTs) isolated from human spinal cord to identify genes containing unstable DNA regions as candidates for disorders of motor function.

Expansion of unstable DNA regions containing trinucleotide/tandem repeats (TNRs) represents a common genetic mutation in hereditary forms of neurological disorders. The spectrum of neurological diseases linked to TNR expansions has recently broadened to include conditions with both dominant and recessive inheritance and those with or without clinical anticipation. In view of the frequent involvement of the spinal cord in neurodegenerative disorders, we have analysed this key tissue to identify pathological TNRs. We have used two approaches to isolate a wide range of trinucleotide/tandem repeat-containing transcripts (TNRTs) from human spinal cord, firstly a polymerase chain reaction (PCR)-based method and secondly by screening a spinal cord cDNA library immobilised on a membrane. Overall, 97 TNRTs belonging to a number of key protein families, the most highly represented being transcription factors, intracellular signalling molecules and cytoskeletal proteins, have been isolated most of which have not previously been considered as potential disease-causing genes. The commonest repeat motifs found in our study were CAG (37%) and CCG (24%). Known genes involved in DNA repeat expansion-related neurological disorders (e.g., AAD10, Ataxin-3, Huntingtin) were detected which validated our methods. We have characterised homogeneous TNRs among the detected gene candidates in a search for potential pathological repeat expansions. The potential role of the gene candidates identified is discussed in terms of their contribution to neurodegenerative processes.

Gene Library↗