Biomedical subjects

T K Das

Publications and source records attributed to T K Das.

At least 37 records · Page 2Linked to original sources

PubMed1999

Redox-linked transient deprotonation at the binuclear site in the aa(3)-type quinol oxidase from Acidianus ambivalens: implications for proton translocation.

The hyperthermophilic archaeon Acidianus ambivalens expresses a membrane-bound aa(3)-type quinol oxidase, when grown aerobically, that we have studied by resonance Raman spectroscopy. The purified aa(3) oxidase, which does not contain bound quinol, undergoes a reversible slow conformational change at heme a(3) upon reduction, as indicated by a change in the frequency of its heme formyl stretching mode, from 1,660 cm(-1) to 1,667 cm(-1). In contrast, upon reduction of the integral membrane enzyme or the purified enzyme preincubated with decylubiquinol, this mode appears at 1,667 cm(-1) much more rapidly, suggesting a role of the bound quinol in controlling the redox-linked conformational changes. The shift of the formyl mode to higher frequency is attributed to a loss of hydrogen bonding that is associated with a group having a pKa of approximately 3.8. Based on these observations, a crucial element for proton translocation involving a redox-linked conformational change near the heme a(3) formyl group is postulated.

Electron Transport Complex IV↗

PubMed1999

Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands.

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.

Amino Acid Sequence↗

PubMed1999

The heme environment in barley hemoglobin.

To elucidate the environment and ligand structure of the heme in barley hemoglobin (Hb), resonance Raman and electron paramagnetic resonance spectroscopic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Barley Hb has a unique heme environment as judged from the Fe-CO and C-O stretching frequencies in the CO complex. Two Fe-CO stretching modes are observed with frequencies at 534 and 493 cm-1, with relative intensities that are pH sensitive. The 534 cm-1 conformer shows a deuterium shift, indicating that the iron-bound CO is hydrogen-bonded, presumably to the distal histidine. A C-O stretching mode at 1924 cm-1 is assigned as being associated with the 534 cm-1 conformer. Evidence is presented that the high Fe-CO and low C-O stretching frequencies (534 and 1924 cm-1, respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm-1 conformer arises from an open conformation of the heme pocket and becomes the dominant population under acidic conditions when the distal histidine moves away from the CO. Strong hydrogen bonding between the bound ligand and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stability to the bound oxygen. This stabilization is confirmed by the dramatic decrease in the oxygen dissociation rate compared with sperm whale myoglobin.

Electron Spin Resonance Spectroscopy↗

PubMed1999

Hydroxide rather than histidine is coordinated to the heme in five-coordinate ferric Scapharca inaequivalvis hemoglobin.

The ferric form of the homodimeric Scapharca hemoglobin undergoes a pH-dependent spin transition of the heme iron. The transition can also be modulated by the presence of salt. From our earlier studies it was shown that three distinct species are populated in the pH range 6-9. At acidic pH, a low-spin six-coordinate structure predominates. At neutral and at alkaline pHs, in addition to a small population of a hexacoordinate high-spin species, a pentacoordinate species is significantly populated. Isotope difference spectra clearly show that the heme group in the latter species has a hydroxide ligand and thereby is not coordinated by the proximal histidine. The stretching frequency of the Fe-OH moiety is 578 cm-1 and shifts to 553 cm-1 in H218O, as would be expected for a Fe-OH unit. On the other hand, the ferrous form of the protein shows substantial stability over a wide pH range. These observations suggest that Scapharca hemoglobin has a unique heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the functional deoxy form of the protein but not in the ferric form.

Animals↗

PubMed1999

Temperature dependent quaternary state relaxation in sol-gel encapsulated hemoglobin.

Samples of human adult hemoglobin (HbA) encapsulated in a wet porous sol-gel are prepared under aerobic and anaerobic conditions. Resonance Raman spectroscopy is used to compare equilibrium deoxyHbA to the nonequilibrium deoxy species generated by deoxygenating an encapsulated oxyHbA sample. The spectra of the deoxygenated samples as a function of delay subsequent to deoxygenation reveal a marked slow down by the gel of the two phases of relaxation: the tertiary relaxation associated with the transition from the liganded R to deoxy R conformations and the quaternary relaxation associated with the deoxy R to deoxy T transition. The temperature dependence (4-80 degrees C) of the relaxation indicates that the internal viscosity of the gel is greatly enhanced at the lower temperatures. At 80 degrees C the tertiary and quaternary relaxations occur over minutes to hours, respectively, whereas at 4 degrees C both relaxations are essentially frozen. These results demonstrate the impressive potential of using sol-gel encapsulation as a means of studying substrate binding induced conformational changes in proteins.

Gels↗

PubMed1999

Pentacoordinate hemin derivatives in sodium dodecyl sulfate micelles: model systems for the assignment of the fifth ligand in ferric heme proteins.

Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.

Biophysical Phenomena↗

PubMed1998

The post-translational modification in cytochrome c oxidase is required to establish a functional environment of the catalytic site.

Mutation of tyrosine-288 to a phenylalanine in cytochrome c oxidase from Rhodobacter sphaeroides drastically alters its properties. Tyr-288 lies in the CuB-cytochrome a3 binuclear catalytic site and forms a hydrogen bond with the hydroxy group on the farnesyl side chain of the heme. In addition, through a post-translational modification, Y288 is covalently linked to one of the histidine ligands that is coordinated to CuB. In the Y288F mutant enzyme, the "as-isolated" preparation is a mixture of reduced cytochrome a and oxidized cytochrome a3. The cytochrome a3 heme, which is largely six-coordinate low-spin in both oxidation states of the mutant, cannot be reduced by cytochrome c, but only by dithionite, possibly due to a large decrease in its reduction potential. It is postulated that the Y288F mutation prevents the post-translational modification from occurring. As a consequence, the catalytic site becomes disrupted. Thus, one role of the post-translational modification is to stabilize the functional catalytic site by maintaining the correct ligands on CuB, thereby preventing nonfunctional ligands from coordinating to the heme.

Binding Sites↗

PubMed1998

Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding.

The mechanism of unfolding of ferricytochrome c induced by the surfactant sodium dodecyl sulfate has been studied by heme absorption, tryptophan fluorescence, circular dichroism, resonance Raman scattering, stopped-flow and time-resolved resonance energy transfer to obtain a comprehensive view of the whole process. Unfolding occurred at an almost specific molecular ratio of SDS/cytochrome c in the concentration range (20-50 microM) studied here. However there appears to be a point at approximately 0.6 mM SDS where unfolding begins to occur for lower cytochrome c concentrations. The kinetics of unfolding revealed only a single transition with a rate constant of 33 s(-1) (at 298 K, [SDS] = 8.7 mM) and activation energy barrier of approximately 16 kJ/mol, indicating that other associated steps, if any, are too fast to be significantly populated. The free energy change (deltaG(o)) involved with the unfolding transition was estimated to be about 16.8 kJ/mol. The CD spectrum at 220 nm of SDS-unfolded cytochrome c shows only a partial decrease (25%), indicating that a significant amount of helical structure remains folded in contrast to a complete loss of helical structure in GdnHCl-denatured cytochrome c. The heme structure in SDS-unfolded cytochrome c, as deduced from heme absorption and resonance Raman spectra, shows a major population (approximately 95%) of mis-ligated histidine to the heme which acts as a kinetic trap in the folding process. The structural changes associated with cytochrome c unfolding were also monitored by time-resolved resonance energy transfer which shows a drastic increase in tryptophan fluorescence lifetime from 12 ps in the native protein to 0.63 ns in the unfolded one, associated with a movement of Trp59 by 10 A away from heme. The maximum entropy method analysis of fluorescence decay indicated the growth of various conformational substates in SDS-unfolded cytochrome c in contrast to narrowly distributed conformations in the native protein. The refolding was comprised of three kinetic steps; the first was significantly fast (approximately 8 ms) and was assigned to the dissociation of His26 that paves the protein towards correct folding pathway. The other two slower steps probably arise from chain misorganization and prolyl isomerization. The absence of a burst-phase amplitude supports the idea that the burst phase observed in the folding from completely unfolded cytochrome c corresponds to a molecular collapse that produces significant secondary structure. The partially unfolded state represents a unique intermediate state in the folding pathway.

Animals↗

PubMed1998

The apolar distal histidine mutant (His69-->Val) of the homodimeric Scapharca hemoglobin is in an R-like conformation.

The effect of the apolar mutation of the distal histidine (His69-->Val) has been studied in the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Absorption, circular dichroism, and resonance Raman spectroscopy point to a more symmetric heme structure of the deoxy derivative, which is indicative of an R-like conformation of the deoxy heme. Resonance Raman spectroscopy also brings out alterations in the geometry and interactions of the bound CO molecule. The iron-carbon stretching frequency is decreased by about 30 cm-1 with respect to the native protein, while the diatomic ligand stretching frequency is increased by about the same degree. Consistent with the structural changes, the ligand binding properties are significantly altered. In the mutant the overall rate and the affinity for CO binding are increased about 100-fold with respect to the native protein, and cooperativity is abolished. In addition, the amplitude and the rate of the geminate rebinding process increase significantly. This finding may be correlated to the longer average residence time of the photolyzed CO molecule within the heme pocket of the H69V mutant, as indicated by molecular dynamics simulations.

Amino Acid Substitution↗

PubMed1998

Effects of smoking cessation on maternal airway function and birth weight.

OBJECTIVE: To evaluate the effects of smoking cessation before or early in pregnancy on maternal airway function and birth weight. METHODS: Measurements of forced expiratory spirometry including forced vital capacity, forced expiratory volume in 1 second, forced expiratory flow rates between 0.2 and 1.2 L, 25% and 75%, and 75% and 85%, and instantaneous flows at lung volumes of 25%, 50%, and 75% were carried out by a wedge bellow spirometer in 40 pregnant exsmokers and were compared with those of 175 nonsmoking and 97 currently smoking pregnant women. Spirometric testing was conducted at a mean (+/- SD) gestational age of 21.5 +/- 7.0 weeks. In exsmokers, the average lifetime cigarette consumption was 17.1 +/- 8.1/day for a mean duration of 9.7 +/- 3.9 years, similar to that observed in current smokers. The median duration of smoking abstinence was 20 weeks before study spirometry. RESULTS: All spirometric measurements in exsmokers were similar to those of nonsmokers and were significantly higher than those of current smokers. Spirometric measurements for nonsmokers, current smokers, and exsmokers were respectively: forced expiratory volume in 1 second (3.36 +/- 0.39, 3.09 +/- 0.45, and 3.35 +/- 0.32 L); forced expiratory flow rate between 25% and 75% (ie, mid-expiratory phase) (3.85 +/- 0.69, 3.21 +/- 0.76, and 3.86 +/- 0.66 L/sec); forced expiratory flow rate between 75% and 85% (ie end-expiratory phase) (1.39 +/- 0.35, 1.03 +/- 0.35, and 1.41 +/- 0.39 L/sec); instantaneous flow at lung volume of 50% (4.35 +/- 0.82, 3.76 +/- 0.89 and 4.36 +/- 0.68 L/sec); and instantaneous flow at lung volume of 25% (1.91 +/- 0.47, 1.47 +/- 0.49, and 1.92 +/- 0.46 L/sec). Mean gestational age at delivery was similar among the three groups (277 +/- 11,274 +/- 12, and 274 +/- 11 days for nonsmokers, current smokers, and exsmokers, respectively). The mean birth weight of babies born to exsmokers (3408 +/- 511 g) was similar to that of babies born to nonsmokers (3469 +/- 461 g), but was significantly greater than that of babies born to smoking pregnant women (3189 +/- 485 g; P < .001). CONCLUSION: Smoking cessation either before or at an early stage of pregnancy is associated with early, reversible increments of maternal airway function and mean birth weights that are higher than among women who continue smoking.

Adult↗

PubMed1998

Antifungal activity of Indian plant extracts.

In vitro studies of water extracts of plants such as Terminalia chebula, Punica granatum, Delonix regia and Emblica officinalis were found to be detrimental to test dermatophytes.

Antifungal Agents↗

PubMed1998

Timing and magnitude of changes in basal energy expenditure during pregnancy in Indian women.

Basal energy expenditure (BEE) was determined in 291 pregnant women, age 20-35 years, using Benedict Roth Metabolism Apparatus. A control study was undertaken in 38 non pregnant women during both follicular and luteal phases of menstrual cycle respectively. The mean +/- SD of BEE were found to be 34.04 +/- 3.05, 35.85 +/- 2.60 and 39.69 +/- 2.75 Kcal/m2/hr during first, second and third trimesters of pregnancy respectively. BEE was progressively and significantly increased (P < 0.01). However, increase in BEE during first trimester of pregnancy compared to that of luteal phase of menstrual cycle was insignificant. The results indicate that Indian pregnant women should maintain energy requirements by increasing caloric intake throughout the gestation.

Adult↗

PubMed1998

Effects of the menstrual cycle on timing and depth of breathing at rest.

Volume and timing components of resting ventilation were measured serially in 40 women aged 18 to 36 yr, during menstrual, follicular and luteal phases of menstrual cycle. Resting minute ventilation (VE) was significantly higher (P < 0.001) in luteal phase than in menstrual and follicular phases; in the two latter phases VE was almost equal. This increment in VE during the luteal phase was due to a significant rise (P < 0.001) in tidal volume (VT). Respiratory frequency (f) was unchanged throughout the cycle. Although there was a mean increases in inspiratory time (T1) during the luteal phase compared to the other two phases, the difference did not reach statistical significance. Duty cycle, T1/Ttot, was also unchanged throughout menstrual cycle. However, mean inspiratory flow, VT/T1, was significantly higher (P < 0.05 and P < 0.01) during luteal phase as compared to that during menstrual or follicular phases respectively. Pulmonary mechanics, as measured by forced vital capacity (FVC), forced expiratory volume in one second (FEV1) and forced mid expiratory flow rate (FEF25%, 75%), were within normal limits and remained unaltered during the menstrual cycle. Therefore, in the absence of alteration of pulmonary mechanics, the luteal increase in ventilation and inspiratory flow suggests a possible role for progesterone in stimulating the respiratory drive, either centrally or through the peripheral chemoreceptors or by both.

Adolescent↗

PubMed1997

Folding of cytochrome c initiated by submillisecond mixing.

Cytochrome c folding was initiated using a new solution mixer that provides a time window which covers over 90% of the burst phase unresolved by conventional stop-flow measurements. Folding was followed by resonance Raman scattering. Kinetic analysis of the high frequency Raman data indicates that a nascent phase occurs within the mixing dead time of 100 microseconds. A significant fraction of the protein was found to be trapped in a misfolded bis-histidine form during the nascent phase at pH 4.5, thereby preventing the protein from folding rapidly and homogeneously. The nascent phase was followed by a haem-ligand exchange phase that populates the native histidine-methionine coordinated form through a thermodynamically controlled equilibrium.

Animals↗

PubMed1997

Primary pulmonary hypertension in non-cirrhotic portal fibrosis.

BACKGROUND: Primary pulmonary hypertension (PPH) has been reported in association with cirrhosis and extrahepatic portal venous obstruction; reports of PPH in noncirrhotic portal fibrosis (NCPF) are few. AIM: To evaluate pulmonary arterial pressure in patients with NCPF. METHODS: Twenty two patients with NCPF underwent hemodynamic studies for pulmonary arterial pressure after excluding secondary causes of pulmonary hypertension. Hemodynamic studies were carried out through the femoral route using 7F Swan-Ganz catheter. Splenoportal venography was done by percutaneous splenic puncture. RESULTS: The mean pulmonary arterial pressure was 12.9 +/- 3.1 mmHg with pulmonary capillary wedge pressure of 8.3 +/- 2.1 mmHg in 20 of 22 cases; in the remaining two cases, the corresponding pressures were 30 mmHg and 28 mmHg and 13 mmHg and 12 mmHg, respectively. CONCLUSION: Two of 22 patients with NCPF had PPH. PPH can thus develop without hepatocellular failure or recurrent embolization from portal axis thrombosis as has been described in cirrhosis.

Adult↗

PubMed1997

Role of circulating immune complexes in prognostic evaluation and management of genitourinary cancer patients.

Circulating immune complexes (CIC) were estimated in 48 Patients of genitourinary cancer by polyethylene glycol precipitation (PEG pptn.) test and latex agglutination inhibition (LAI). The results were compared with 25 healthy control volunteers. Pathological levels of CIC were observed in 79.18 percent patients of genitourinary cancer by combination of PEG pptn. and LAI tests, while no seropositivity for CIC was observed in control group (p < 0.001). Sequential increase in seropositivity for CIC was observed with advancing stage of genitourinary cancer i.e. number of seropositive patients in cancer stage I were 60 percent, stage II-71.42 percent, stage III-85.71 percent and stage IV-100 percent. Variation IN CIC levels in different patients within the same stage are compared. Circulating antigen antibody complexes have a significant role as prognostic monitors in management of genitourinary cancer patients. Statistical evaluation of data on intra- and inter-assay variation has been given. CIC levels rise with increases in tumor burden in vivo hence variation in CIC levels within the same stage in different patient have a significant role as prognostic monitors in management of individual patients.

Adult↗

PubMed1996

An M. tuberculosis DNA fragment contains genes encoding cell division proteins ftsX and ftsE, a basic protein and homologues of PemK and small protein B.

A 4-kb fragment of the M. tuberculosis chromosome was identified which contains several genes including those involved in cell division and possibly macrophage survival. DNA sequence analysis revealed open reading frames (ORFs) encoding putative proteins bearing significant homology with proteins FtsX and FtsE associated with cell division in E. coli, with PemK protein which inhibits cell division in E. coli harboring plasmid R100 and with SmpB protein of Salmonella typhimurium implicated in its survival within macrophages. The ftsX gene is conserved among mycobacteria belonging to the M. tuberculosis Complex. Furthermore, ftsX-specific transcripts were prevalent in equivalent amounts in M. tuberculosis H37Rv and H37Ra as analyzed by RT-PCR and primer extension. Transcription start points (tsp) a and b map in the region upstream of the FtsX ORF whose promoter activity was established by (i) a promoter-fusion experiment and (ii) by mapping the 5' ends of transcripts derived from the promoter-fusion construct. FtsX transcription is modulated as a function of mycobacterial growth and division status, maximum expression being observed in log phase cells. Growth-related expression of ftsX may provide a basis for developing a marker to distinguish actively replicating M. tuberculosis cells from quiescent mycobacteria.

ATP-Binding Cassette Transporters↗

PubMed1996

Gastroduodenal manifestations in patients with skeletal fluorosis.

A prospective case-controlled study was performed to evaluate the gastrointestinal symptoms and mucosal abnormalities occurring in patients with osteofluorosis. Ten patients with documented osteofluorosis and ten age- and sex-matched healthy volunteers were included in the study. Clinical evaluation, real-time ultrasound, and upper gastrointestinal endoscopy and biopsy from the gastric antrum and duodenum were performed in all subjects. The biopsies were subjected to a rapid urease test and light and electron microscopic examinations. Ionic fluoride levels were estimated in the drinking water, serum, and urine using an ION 85 ion analyzer. All patients with osteofluorosis had gastrointestinal symptoms, the most common being abdominal pain. Endoscopic abnormalities were found in seven patients with osteofluorosis. In all 7 of these patients, chronic atrophic gastritis was seen on histology. Electron microscopic abnormalities were observed in all 10 patients with osteofluorosis. These included loss of microvilli, cracked-clay appearance, and the presence of surface abrasions on the mucosal cells. None of the control subjects had any clinical symptoms or mucosal abnormalities. It was concluded that gastrointestinal symptoms as well as mucosal abnormalities are common in patients with osteofluorosis.

Abdominal Pain↗