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V Kohli

Publications and source records attributed to V Kohli.

50 records · Page 3Linked to original sources

Ribozymes that cleave an RNA sequence from human immunodeficiency virus: the effect of flanking sequence on rate.

Ribozymes designed to cleave sequences specific to viral RNA may be better antiviral agents than simple antisense oligonucleotides. High catalytic activity with the lowest possible chain length is desired for this purpose. We have synthesized several hammerhead ribozymes that cleave sequences from HIV-1 RNA. On reducing from 20 to 12 the base pairs formed with the substrate, the rate of cleavage at 37 degrees C increased 10-fold. Deletions from the stem/loop structure in the ribozyme also increased the initial rate of reaction.

Base Sequence↗

D-penicillamine.

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Child↗

Resistance to 4-(9-acridinylamino) methanesulphon-m-anisidide (m-AMSA) in human myeloid leukaemia.

Sublines of a human myeloid leukaemia cell line, KBM-3, with increasing degrees of resistance to the antileukaemic agent 4'-(9-acridinlylamino) methanesulphon-m-anisidide (m-AMSA) were evaluated for their response to this drug using a clonogenic assay to measure cell survival and alkaline elution to assess m-AMSA induced DNA strand breakage. Polyacrylamide gel electrophoresis was used to map the protein profiles of the various cell lines. The resistant lines were obtained by intermittent exposure of the KBM-3 cells to the highest tolerated concentration of m-AMSA so that the culture would be repopulated only by the most resistant subpopulation after each exposure. Two distinct phases were apparent during the development of resistance. During the first 14 months of intermittent exposure to maximally tolerated concentrations of m-AMSA, the cells developed low-degree m-AMSA resistance (5-7-fold as compared with the parent line, as measured by cell survival). This low-degree resistance was characterised by a somewhat suppressed level of DNA strand breakage and no measurable change in cellular protein levels. Subsequently, a single escalation of the m-AMSA retreatment concentration resulted in a cell population that was approximately 100-fold resistant, as assessed by cloning. This rapid phenotypic change temporally coincided with the acquisition of an almost complete refractoriness to m-AMSA-induced DNA strand breakage and the loss of a cellular 76 kDa protein. We suggest that the loss of this protein is important for the development of a highly m-AMSA resistant phenotype.

Amsacrine↗

High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli.

A cDNA clone containing the complete human alpha 1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe. DNA sequences encoding the alpha 1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage lambda (PL) and initiation of translation at the lambda cII gene ribosome-binding site. This construction resulted in the induction of a 45-kilodalton protein at a level of approximately 15% of total cell protein. The polypeptide produced was recognized by antisera raised against human alpha 1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay.

Amino Acid Sequence↗

Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.

A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

Alanine↗

Silica gel: an improved support for the solid-phase phosphotriester synthesis of oligonucleotides.

The phosphotriester method for the stepwise synthesis of deoxyoligonucleotides has been employed using HPLC-grade silica gel (Porasil B) as the solid support. The procedure results in a convenient flow-through system for the synthesis of oligomers where all the reaction steps including the zinc bromide method of detritylation are compatible with the selected support. Deoxyoligonucleotides of 25-30 nucleotides in length can be synthesized in high yields utilising stable phosphotriester intermediates. Ease of handling of the solid support allows convenient synthesis of mixed oligonucleotide sequences.

Base Sequence↗

Fusion of restriction termini using synthetic adaptor oligonucleotides.

A method is proposed for linking a DNA fragment possessing a 5' single-stranded extension to one carrying a 3' extension. Synthetic oligonucleotide adaptors can be used to (i) change the site specificity at the termini of a fragment generated by restriction enzyme cleavage and (ii) simultaneously dephosphorylate the extremities of a DNA molecule to prevent recircularisation and allow positive selection for recombinant DNA molecules.

Amino Acid Sequence↗

Some improvements in the synthesis of DNA of biological interest.

In a brief summary the improvements introduced during synthesis of a minigene coding for the peptide hormone angiotensin II are outlined. Furthermore some recent improvements are reviewed: comprehensive study of N-protecting groups for the heterocyclic bases, effective 3'-O-phosphorylation using phosphomonochloridates and molecular sieves, a quick and mild detritylation procedure without any depurination using ZnBr2, a convenient tritylation method using molecular sieves, introduction of a sugar spray reagent for a simpler interpretation of tlc, an improved two-step-one-flask procedure for the synthesis of fully protected triester intermediates, an introduction of new phosphate protecting groups removable by beta-elimination in homogeneous phase, synthesis of the internucleotidic phosphotriester linkage using phosphomonoazolide derivatives of deoxynucleosides, versatile methods for anchoring nucleosides to a polymer support and a computerized nucleic acid synthesizer.

Angiotensin II↗

Coronary artery bypass surgery without cardiopulmonary bypass: short- and mid-term results.

From March 1994 to April 1997, 433 patients had undergone coronary artery bypass grafting without cardiopulmonary bypass in our institute. Sixty-eight patients had various organ dysfunctions and/or aortic atheroma or calcification and were regarded as high risk for cardiopulmonary bypass. In 277 patients surgery was performed through midline sternotomy, while in 156 minithoracotomy approach was used. In 361 patients single coronary artery bypass grafting was done, and in 72 two-coronary arteries were bypassed. In 63 patients who had graftable vessels in anterior wall and diffusely diseased ungraftable vessels in posterolateral and/or inferior wall, transmyocardial laser revascularisation was also done along with coronary artery bypass grafting to achieve complete myocardial revascularisation. Nine patients in this series were also subjected to simultaneous carotid endarterectomy along with myocardial revascularisation. In two patients complementary percutaneous transluminal coronary angioplasty of left circumflex coronary artery was done five days after minithoracotomy and left internal mammary artery to left anterior descending coronary artery bypass grafting. Forty-two cases were extubated in operating room. Average blood loss was 260 ml. Six patients were reexplored for postoperative bleeding. Seven patients had perioperative myocardial infarction. One developed neurological complication. Hospital mortality was 2.3 percent (10/433 cases) and four deaths were due to malignant ventricular arrhythmias. Nine patients developed chest wound complications. Average hospital stay after operation was six days, 423 patients were discharged from hospital and all of them were asymptomatic. During three years follow-up (range 3 to 38 months) there were three known cardiac deaths. Ninety percent (391) patients reported to the follow-up clinic and 91 percent of them were angina-free. In patients who were subjected to transmyocardial laser revascularisation along with coronary artery bypass grafting, myocardial perfusion scan showed a step-wise improvement in reversible ischemia. The perfusion index increased from 52 percent at three months to 90 percent at 12 months. We conclude that coronary artery bypass grafting without cardiopulmonary bypass can be done with relatively low mortality, more so in a group of patients in whom cardiopulmonary bypass poses a high risk. Transmyocardial laser revascularisation is a suitable means to provide complete myocardial revascularisation along with coronary artery bypass surgery in patients who have graftable vessels in anterior wall and ungraftable vessels in posterolateral and inferior walls.

Adult↗