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Biomedical subjects

A K Basu

Publications and source records attributed to A K Basu.

At least 19 recordsLinked to original sources
PubMed1992

Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.

We previously described a protein, isolated from human tissues and cells, that bound to a defined double-stranded oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine. It was further demonstrated that this protein was a glycosylase and released 1,N6-ethenoadenine. We now find that this enzyme also releases 3-methyladenine from methylated DNA and that 3-methyladenine-DNA glycosylase behaves in the same manner, binding to the ethenoadenine-containing oligonucleotide and cleaving both ethenoadenine and 3-methyladenine from DNA containing these adducts. The rate and extent of glycosylase activities toward the two adducts are similar.

Adenine↗
PubMed1989

Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies.

The mutational specificity and genetic requirements for mutagenesis by 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), one of the principal DNA lesions induced by oxidation and ionizing radiation, has been investigated in Escherichia coli. Thymine glycol was positioned at a unique site in the single-stranded genome of a bacteriophage M13mp19 derivative. Replication of the genome in E. coli yielded targeted mutations at a frequency of 0.3%; the mutations were exclusively T----C. Mutagenesis was independent of SOS and nth (nth encodes endonuclease III, a thymine glycol repair enzyme). The adduct was not detectably mutagenic in duplex DNA. A chemical rationalization for the mutation observed for thymine glycol was developed by applying molecular modeling and molecular mechanical calculations to the same DNA sequence studied in vivo. Modeling suggested that the 5R,6S isomer of cis-thymine glycol, when not base paired, was displaced laterally by approximately 0.5 A toward the major groove in comparison to the position that thymine would otherwise occupy. This perturbation of DNA structure should increase the likelihood of a guanine.thymine glycol wobble base pair during replication, which would explain the mutational specificity of the base observed in the genetic experiments.

Base Sequence↗
PubMed1989

Site-specific mutagenesis by O6-alkylguanines located in the chromosomes of mammalian cells: influence of the mammalian O6-alkylguanine-DNA alkyltransferase.

A plasmid was constructed in which a single guanine residue was replaced with either O6-methylguanine or O6-ethylguanine, two of the DNA adducts formed by carcinogenic alkylating agents. The vectors were introduced in parallel into a pair of Chinese hamster ovary cells, in which one member of the pair was deficient in the repair enzyme O6-alkylguanine-DNA alkyltransferase (mex-) and the other was proficient in this activity (mex+). The vectors integrated into and replicated within the respective host genomes. After intrachromosomal replication, the DNA sequence in the vicinity of the originally adducted site of each integrated vector was amplified from the host genome by using the polymerase chain reaction and was analyzed for mutations. High levels of mutation were observed from the O6-methylguanine- and O6-ethylguanine-containing vectors replicated in mex- cells (approximately 19% for O6-methylguanine and approximately 11% for O6-ethylguanine). DNA sequencing revealed the induced mutations to be almost exclusively G----A transitions. By contrast, little or no mutagenesis was detected when the adducted vectors were introduced into mex+ cells, indicating the significant role of the O6-alkylguanine-DNA alkyltransferase in the repair of O6-methylguanine and O6-ethylguanine in these mammalian cells.

Animals↗
PubMed1989

Atenolol for the prevention of arrhythmias following coronary artery bypass grafting.

Seventy patients undergoing aortocoronary bypass grafting were randomized, double-blind, to receive either atenolol or placebo. There were 35 patients in each group. Patients received either atenolol 5 mg intravenously or matching placebo within 3 h of the completion of surgery. A second intravenous dose was administered 24 h following the first and then atenolol 50 mg orally or matching placebo was given for six days. Continuous Holter monitor recordings were obtained for the 24 h immediately preoperatively and continuously for eight days postoperatively. No patient received any antiarrhythmic drug preoperatively. Patients who required pharmacological intervention for the management of postoperative arrhythmias were withdrawn as treatment failures. Holter monitor analysis continued for 24 h following withdrawal of a treatment failure. All patients were analyzed according to the intention-to-treat principle. Both groups were comparable with respect to age, sex, severity of coronary artery disease, left ventricular ejection fraction, preoperative use of beta-blockers, bypass time, aortic cross-clamp time, number of grafts per patient and frequency of preoperative arrhythmias. Arrhythmia analysis was done manually. Supraventricular arrhythmias (atrial tachycardia, atrial fibrillation and atrial flutter) were classified as either mild (less than 0.5 mins, less than 140 beats/min), moderate (0.5 to 30 mins, 140 to 180 beats/min), or severe (longer than 30 mins, more than 180 beats/min). Ventricular arrhythmia analysis was performed with respect to isolated PVCs, couplets, triplets and episodes of nonsustained ventricular tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1989

Mutagenic specificity of alkylated and oxidized DNA bases as determined by site-specific mutagenesis.

This work demonstrates the use of the tools of site-specific mutagenesis to study the mutagenic activity of two DNA adducts, O6-methylguanine and cis-thymine glycol. The former adduct is one of the methylated bases formed by carcinogenic and mutagenic alkylating agents. It was built into the single-stranded genome of bacteriophage M13 and replicated in Escherichia coli (E. coli). The mutation frequency of O6-methylguanine was 0.4% in physiologically normal cells. In cells in which the repair systems for O6-methylguanine were compromised by challenge with an alkylating agent, the mutation frequency rose to approximately 20%. DNA sequencing revealed that O6-methylguanine induced exclusively G----A transitions, which was most consistent with it pairing with thymine during DNA synthesis. The mutagenic effects also were investigated of cis-thymine glycol isomers, which are major, stable products of ionizing radiation and oxidative damage to DNA. By techniques similar to those employed for the study of O6-methylguanine mutagenesis, a single thymine glycol was situated in an M13 phage genome. The genome was replicated in E. coli that were physiologically normal, induced for SOS functions, or deficient in the nth gene product and, in all cases, the mutagenic processing of thymine glycol in vivo yielded mutant progeny phage at a frequency of 0.3-0.4%. All mutations occurred at the site that originally contained thymine glycol, and all were demonstrated by DNA sequencing to have resulted from targeted T----C transitions. These data suggest that thymine glycol pairs with guanine during replication.

DNA Mutational Analysis↗
PubMed1989

Significance of transplant perfusion index in the diagnosis of acute rejection in live related renal allograft recipients.

The study was undertaken to assess the usefulness of transplant perfusion index (TPI) in the differential diagnosis of renal allograft dysfunction with special reference to acute rejection. It was observed that the TPI has a sensitivity of 100 per cent and specificity of 98.1 per cent in the diagnosis of acute rejection. It was also observed that the serial values of TPI provided valuable clues and guide in the management of transplant dysfunction and helped in the immediate and long term follow-up of patients with renal allografts.

Adult↗
PubMed1988

O6-alkyldeoxyguanosine detection by 32P-postlabeling and nucleotide chromatographic analysis.

The 32P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quantitation of O6-alkyl-2'-deoxyguanosine residues in DNA. Chromatographic techniques were developed to resolve individually the normal deoxyribonucleotide-3'-monophosphates and the O6-alkyldeoxyguanosine-3'-monophosphates by high-pressure liquid chromatography. Selective deoxyribonucleotide-3'-monophosphates (e.g., O6-alkyldeoxyguanosine-3'-monophosphates) were then converted to labeled deoxyribonucleotide-[5'-32P]monophosphates by 32P-postlabeling and nuclease P1 treatment and separated by two-dimensional thin layer chromatography. The O6-methyl- and O6-ethyl-2'-deoxyguanosine-3'-monophosphate nucleotides, and the respective 5'-monophosphates, were chemically synthesized for standardization of these quantitative procedures. The quantitation of O6-methl- and O6-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O6-alkyl-2'-deoxyguanosine residue per 10(4) and 10(7) 2'-deoxyguanosines. The limit of detection was less than one O6-alkyl-2'-deoxyguanosine in 10(7) 2'-deoxyguanosine residues in a sample size of 100 micrograms of DNA, i.e., approximately 10 pg of adduct. The quantitation of O6-methyl-2'-deoxyguanosine in the liver DNAs of rats treated with [14C-Me]N-nitrosodimethylamine compared well with values obtained by both 14C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these 32P-postlabeling and nucleotide chromatographic procedures should be useful in monitoring human exposure to methylating and ethylating carcinogens.

Alkylation↗
PubMed1987

Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N6-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon.

Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d[GCT(epsilon A)GC] was chemically synthesized by the phosphotriester method. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the epsilon Ade adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique SmaI site of M13mp19. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer [5'-32P]d-[GCT(epsilon A)GC], after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The adduct was introduced into a unique NheI site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.

Adenosine↗
PubMed1987

A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site.

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5'-d(TpG8-ABPpCpA)-3' and 5'-d(TpGpCpA)-3' were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease PstI, in an otherwise duplex genome of M13mp10. In the case of the adducted tetranucleotide, dG8-ABP was located in the minus strand at genome position 6270. Experiments in which the tetranucleotides were 5' end labeled with [32P]phosphate revealed the following: the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. Evidence also is presented showing that the dG8-ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminobiphenyl Compounds↗
PubMed1987

Marrow uptake index (MUI): a quantitative scintigraphic study of bone marrow in aplastic anaemia.

Aplastic anaemia affects the entire bone marrow. Current methods of assessment of bone marrow function, like bone marrow biopsy or peripheral blood examination are either invasive or inadequate and cannot be expected to represent fully the changes in the entire bone marrow tissue. This prospective study was undertaken to develop and standardise a new Nuclear Medicine technique called 'Dynamic Bone marrow Imaging'. Eleven patients and ten controls were studied. Serial images of the pelvis were obtained in frame mode following intravenous injection of 185-370 mBq of 99mTc S. Colloid, and an index, called the Bone Marrow Uptake Index (P) was calculated by taking into consideration the time activity curve obtained over the iliac crest. This was followed by static imaging of the entire bone marrow in all cases. It was possible to obtain excellent information regarding topographic distribution of bone marrow as well as detect early changes in bone marrow function following treatment. An attempt was also made to correlate bone marrow cellularity as obtained by bone marrow biopsy with the results of dynamic bone marrow scintigraphy. On the basis of the encouraging results obtained in the present study, the authors feel that dynamic bone marrow imaging is an excellent technique for the objective evaluation of bone marrow in aplastic anaemia. Aplastic anaemia affects the entire bone marrow tissue. Although much progress has been made in the management of this disease, many aspects of it await better understanding. There is almost total lack of knowledge regarding the distribution of functioning marrow in various phases of aplastic anaemia, such as in relapse and remission. Current methods of assessment of marrow function rely mainly on bone marrow biopsy and peripheral blood examination. Bone marrow biopsy is invasive and cannot be expected to represent fully the changes in the entire tissue. Changes in peripheral blood picture lag behind the changes in the bone marrow. Thus, there is a need for an investigation which is safe, simple, sensitive, non invasive and capable of assessing the global function of bone marrow. Radio-nuclide imaging of bone marrow requires labelling of one or more components of this widely dispersed tissue. The reticuloendothelial and erythropoietic components can be labelled with radio-colloids and radio-iron respectively. Experimental studies have shown that the reticuloendothelial and erythropoietic elements are invariably found together in the marrow and have similar distribution. This report is based on a prospective study of bone marrow function in patients with aplastic anaemia, using 99mTc. Sulphur colloid.

Adolescent↗
PubMed1987

Serendipity in technetium-99m DTPA renal dynamic study.

A nine-year-old child was referred for a routine renal dynamic study to detect a renal and/or renovascular cause for hypertension. Tc-99m DTPA renography accidentally discovered and correctly localized a pheochromocytoma.

Adrenal Gland Neoplasms↗