AIDS and the gateway of the body.
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AIDS-Related Opportunistic Infections↗
Biomedical subjects
D Bandyopadhyay
Publications and source records attributed to D Bandyopadhyay.
Reaction of ferrous cytochrome c peroxidase with dioxygen: site-directed mutagenesis provides evidence for rapid reduction of dioxygen by intramolecular electron transfer from the compound I radical site.
The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a FeII-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23 degrees C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen.(ABSTRACT TRUNCATED AT 250 WORDS)
Cross-Linking Reagents↗
Quaternary structure and geminate recombination in hemoglobin: flow-flash studies on alpha 2CO beta 2 and alpha 2 beta 2CO.
The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1-2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.
Chromatography, High Pressure Liquid↗
Correlations between c-erb beta-2 oncogene amplification and the expression of its mRNA and protein in human breast carcinomas.
Nineteen human breast tumours were analysed for c-erb beta-2 gene amplification, the over-expression of its mRNA and the production of its protein residue by southern blot, northern blot and western blot technique respectively. Protein expression was also assessed by immunohistochemistry on paraffin sections, c-erb beta-2 gene amplification was found in 6 of 19 (31%) tumours. Over-expression of c-erb beta-2 mRNA was found in 7 of 19 (37%) tumours. Expression of the 185 kd c-erb beta-2 protein product was detected in 3 of 19 (16%) tumours by western blotting and in 5 of 19 (26%) by immunohistochemistry. In only 4 tumours with an amplified c-erb beta-2 gene was there a clear association between all three parameters. In view of the conflicting reports in the literature concerning c-erb beta-2 gene amplification or protein over-expression (assessed by western blot or immunohistochemistry) and prognosis of breast cancer, studies in which these parameters are correlated individually with prognosis in the same group of patients are needed.
Breast Neoplasms↗
Double-mixing kinetic studies of the reactions of monoliganded species of hemoglobin: alpha 2(CO)1 beta 2 and alpha 2 beta 2(CO)1.
The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).
Carbon Monoxide↗
Performance characteristics of a commercial dedicated contamination counter.
The dedicated wipe test counter (DWTC) is a commercial geiger-counter type instrument especially designed for wipe test counting. All samples are counted under the same physical conditions within 0.3 cm from a 2-mg cm-2 mica end-window geiger tube of 1.27 cm in diameter. The counting capabilities of the DWTC were tested for different radioisotopes in common use in the nuclear medicine laboratory. Since most of the imaging agents are 99mTc-based radiopharmaceuticals, an experimental efficiency factor, J = 3 cpm kdpm-1, was determined for this radioisotope by the manufacturer and stored in the counting system for default use. In addition, the formulation for the counting time needed to achieve a measurement with adequate level of significance (two standard deviations) and a background measurement time of 20 min was programmed in the firmware of the DWTC. With these parameters, the counting time for a 99mTc wipe (threshold of 2000 dpm) becomes 1.88 min for a background level of 10 cpm. The counting time increases to 3.25 and 4.80 min for the respective background levels of 20 and 30 cpm while the threshold is kept the same. In practice, we were able to measure activities as low as 0.7 kdpm for 99mTc and 0.09 kdpm for 131I. Linearity was maintained for a wide range of activities for all tested radioisotopes. The DWTC was found to be simple to operate and satisfies all requirements for performing wipe tests in the nuclear medicine laboratory.
Evaluation Studies as Topic↗
Isolation and cloning of Bacillus thuringiensis var Kurstaki HD73 toxin gene and construction of a chimaeric gene for expression in plants.
Necessity for the production of transgenic crop plants of India, capable of expression of insecticidal Bt protein in plant to combat lepidopteran pest damage has been strongly felt. Bacillus thuringiensis Kurstaki HD73 crystal protein coded by CryIA(c)73 gene has been found to be sufficiently effective against the major pests of jute and chickpea. An attempt to isolate the gene and make its use in a chimaeric gene construct for expression in plant was carried out. The plasmid CryIA(c)73 gene was cloned and tailored at the 3' end. The expression of the truncated gene was monitored in the minicell systems of E. coli. The entomocidal property was found to be fully retained by the gene product. Deletion of the nucleotides at the 5' end was carried out and chimaeric gene construct of cryIA(c)73 was made in such a way that it was fused in frame with GUS gene under the control of the caMV 35S promoter with Nos polyadenylated terminus. Such a chimaeric gene construct was used as the passenger of a Ti plasmid derived plant vector with kanamycin gene (NPTII) as the additional plant marker. Transformation through infection of tobacco and mustard plant cells in culture was carried out. Plants regenerated from the transformed cells showed the presence of GUS gene indicating the expression of the cloned fused gene. Also, Northern analysis established the presence of cryIA(c)73 gene transcripts in the transgenic plants.
Bacillus thuringiensis↗
Double-mixing kinetic studies of the reactions of methyl isocyanide and CO with diliganded intermediates of hemoglobin: alpha 2CO beta 2 and alpha 2 beta 2CO.
Kinetics of the reactions of CO and methyl isocyanide with two diliganded intermediates of hemoglobin, alpha 2CO beta 2 and alpha 2 beta 2CO, have been studied by double-mixing and microperoxidase methods. The valency hybrids were prepared by high-pressure liquid chromatography. The reaction time courses of ligand combination and dissociation with both of the ligands were biphasic, and in CO combination reaction the zero-time amplitudes of the two phases were independent of the protein concentration. In the presence of 2 M urea the reaction time course was clearly dependent on protein concentration, as the zero-time amplitude of the fast phase increased at lower protein concentrations. These two observations indicate that little dissociation of tetramers into dimers occurs in the absence of urea. Consistent with this, the kinetic data for the reactions of CO best fit a reaction model consisting of two tetrameric species not in rapid equilibrium with each other. Various considerations, however, suggest that the reaction model is more appropriately described as 2D in equilibrium R in equilibrium T. The reaction of triliganded species (Hb4(CO)2Me1) with methyl isocyanide was monophasic, and the reaction model suggested a fast T in equilibrium R structural change after the binding of the third ligand. Although the precise structural nature of the two species remains undefined, it is concluded that the biphasicity in the reactions of the two hybrids is characteristic of the diliganded species only and is independent of the nature of the ligand.
Carbon Monoxide↗
Quaternary structure and the geminate recombination of carp hemoglobin with methylisocyanide.
The kinetics of geminate recombination were studied for the methylisocyanide derivative of carp hemoglobin. Carp hemoglobin is of interest because it has been established that the fully liganded form switches between a high affinity R state at pH 9 and a low affinity T state at pH 6 in the presence of IHP. Geminate recombination was observed on both the picosecond and the nanosecond time scales under all conditions; however, only a small variation is observed in the rates and the yields of geminate recombination as the protein switches from the R to the T state. Taken together with overall "on" and "off" rates, the data indicate that the change from the R to the T configuration affects bond breaking most, but also influences subsequent escape from the protein as well as both entry into the protein and bond formation. There is some reason to postulate tertiary conformational change in the T state on the microsecond time scale following ligand escape from the protein.
Allosteric Regulation↗
Charge-symmetry breaking in np elastic scattering at 477 MeV.
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Journal Article↗
Biodistribution of indium 111-labeled dihematoporphyrin ether in papillomas and body tissues. Relevance to photodynamic therapy.
Hematoporphyrin derivative and its newly purified form, dihematoporphyrin ether, have been shown to localize selectively in malignant tissues and virally induced papillomas. Its use as a probe to distinguish tumors from normal tissues has been largely based on its fluorescence when activated by UV light. These findings are largely subjective, and a direct correlation to its use as a photosensitizing agent to selectively kill transformed cells when activated by an appropriate wavelength of light could not be made. The efficacy of dihematoporphyrin ether photodynamic therapy to selectively kill papillomavirus-transformed cells is based on the increased localization of dihematoporphyrin ether within these tissues as compared with normal tissues. Using cottontail rabbit papillomavirus, cutaneous papillomas were induced on the backs of Dutch belted rabbits. Dihematoporphyrin ether was labeled with indium 111 and intravenously injected into the rabbits. The animals were scanned twice daily for indium 111 activity on a large-field-of-view gamma camera. At 50 hours after injection, the rabbits were killed and papillomas, skin, and major organs collected for biodistribution studies. The results of this study and their relationship to dihematoporphyrin ether photodynamic therapy for the treatment of virally induced papilloma disease is discussed.
Animals↗
np elastic scattering analyzing power characteristics at intermediate energies.
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Journal Article↗
Spin correlation parameter and analyzing power in n-p elastic scattering at intermediate energies.
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Journal Article↗
Ratio of spin transfer parameters dt/rt in 2H(p
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Journal Article↗
Potential use of indium-111-labeled polymorphonuclear leukocytes for the detection of lung microvascular injury.
The early stages of microvascular injury are often difficult to detect due to the lack of a suitable marker to assess such an injury. We utilized the well known phenomenon of polymorphonuclear leukocyte (PMN) migration to the microvascular bed as a result of acute inflammatory reactions originating from the damaged cells. A radiotracer technique was developed, employing indium-111-labeled PMN for the detection of microvascular injury induced by hyperoxia. New Zealand white rabbits exposed to either 100% oxygen or air for various intervals of time were injected with indium-111-tropolone or oxine-labeled PMNs. Influx of radioactive PMN into the lung was detected in 72 hr/oxygen-exposed animals using gamma scintigraphic technique. Analysis of dry/wet ratios and histological examinations of the lung biopsies indicated noncardiogenic edema formation at this stage. Mortality was 50% beyond 96 hr/oxygen exposure. Our study thus provided a means to detect early microvascular injury during 72 hr/oxygen-exposure, which was not detectable by any other noninvasive techniques. The use of indium-111-labeled PMN thus appears to be a potentially important tool for the clinical assessment of lung microvascular injury.
Animals↗
Role of polymorphonuclear leukocytes in hyperoxic lung injury. Prevention of neutrophil influx into the lung endothelium during oxygen exposure by ibuprofen.
The present study examines the role of PMN in hyperoxic lung injury using a nonsteroidal anti-inflammatory drug, ibuprofen. New Zealand white rabbits were exposed to 100% oxygen or air for 1 to 4 days. The animals were divided into two groups: the treatment group received ibuprofen with drinking water, and the control group received water alone. After exposure to oxygen or air, the rabbits were injected with indium-III-oxine labeled PMN. Influx or radioactive PMN into the lung was detected using gamma scintigraphic technique only in 72 h and 96 h oxygen-exposed animals which were not treated with ibuprofen. Ibuprofen-treated animals did not exhibit any PMN influx into the lung. Blood gas analysis of all 72 h oxygen-exposed animals did not show any abnormal values, although analysis of dry/wet weight ratios and histopathological examinations indicated noncardiogenic edema formation. After 96 h of oxygen exposure, both ibuprofen-treated and untreated groups suffered from severe acidosis. Gross cytoplasmic edema and partial destruction of lung endothelium were also observed. Mortality was 50% after 96 h of oxygen exposure. Our study, thus, demonstrates that ibuprofen cannot prevent hyperoxic lung injury although it inhibits the influx of PMN into the injured lung, suggesting that PMN are not directly involved in the injury process.
Animals↗
111Indium-tropolone labeled human PMNs: a rapid method of preparation and evaluation of labeling parameters.
Pure polymorphonuclear leukocytes (PMNs) have been isolated from a small amount of human blood by a single-step density gradient centrifugation method using a commercially available Ficoll-Hypaque mixture of density 1.114. The cells were labelled with [111In]tropolone in both buffer and plasma. Cell viability, ability to generate superoxide anion, and chemotaxis were found to be unaltered both before and after labeling. The optimum tropolone concentration for labeling was found to be 1 X 10(-4) M. Labeling efficiency was higher at 37 degrees C than at room temperature. Compared to [111In]oxine, tropolone preparation both in buffer and plasma resulted in consistently higher yields. Preliminary experiments of in vivo cell viability of the labeled PMNs were carried out in rabbits. The ability of the cells to localize in experimentally produced inflammatory lesions was found to be intact. The method of cell separation and labeling described has been found to be simple and rapid and could easily be incorporated in routine nuclear medicine laboratory practice.
Animals↗
Use of a gamma camera to estimate bone mineral content based on the dual-photon technique.
Bone mineral content evaluations using a point source of 153Gd of strength 100 mCi or less and a Siemens large field of view gamma camera equipped with a converging collimator is reported. Both 44 and 100 ke V images are digitised in 128 x 128 matrices and processed in a Siemens MicroDelta System attached to a VAX 11-750 computer. Shorter imaging time, low source strength and the usage of existing nuclear medicine equipment are the major highlights of the technique. The effective focal distance (EFD) of the collimator used is experimentally determined with a 57Co source and found to be 75 cm. Focal distances are also calculated from measured values of magnification factors at different points on the collimator face to assess the degree of misalignment of converging holes. Due to the high count rate of the system, corrections for dead time losses and image shrinkages are applied. Two additional low-activity point sources of 153Gd, which serve as live time meters of the system, are used for this purpose. In addition, due to the non-homogeneity of the converging collimator, uniformity corrections for each field of view are found to be essential. In external standards made of various amounts of hydroxyapatite, a correlation coefficient greater than 0.990 with a standard error of the estimate (SEE) 0.0225 is obtained between measured and actual values. In a limited clinical trial, high-resolution images of human spines are acquired and quantitative estimates of bone mineral content are evaluated.
Bone and Bones↗