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P Khanna

Publications and source records attributed to P Khanna.

At least 19 recordsLinked to original sources
PubMed2006

Outcome predictors for non-invasive positive pressure ventilation in acute respiratory failure.

BACKGROUND: Noninvasive positive pressure ventilation (NIPPV) has emerged as a significant advancement in the management of acute respiratory failure. OBJECTIVE: To identify factors, based on clinical and laboratory parameters, for predicting the outcome of NIPPV in patients with acute respiratory failure. MATERIALS AND METHODS: Fifty patients were included in the study. Inclusion criteria were RR>30 breaths/ min, PaO2<60mmHg, PaO2/FiO2<300, pCO2< or = 45mmHg and signs of increased work of breathing. Baseline clinical parameters and arterial blood gas (ABG) were recorded before initiating NIPPV. Clinical parameters including heart rate, respiratory rate, oxygen saturation and ABG was revaluated at 1, 4, 12, 24 hrs after initiation of NIPPV. Change in these parameters and need for intubation was evaluated. RESULTS: Of the 50 patients, 37 (74%) showed clinical and ABG improvement. Out of 13 (26%) patients who failed to respond, 7 (52%) needed endotracheal intubation within 1 hr. There was significant improvement in clinical and ABG parameters within 1st hr in success group and these parameters continues to improve even after 4 hrs of NIPPV treatment (p<0.05). Failure group had higher baseline heart rate than success group (p<0.05). CONCLUSION: Determination of baseline clinical factors such as heart rate and respiratory rate, available at the time of initiation and after a short period, can predict the likelihood of success or failure of NIPPV. As a result, delay in intubation can be avoided which itself is associated with significant mortality.

Acute Disease↗
PubMed2001

Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase.

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.

Acetates↗
PubMed2001

Characterization of the FAD-containing N-methyltryptophan oxidase from Escherichia coli.

N-Methyltryptophan oxidase (MTOX) is a flavoenzyme that catalyzes the oxidative demethylation of N-methyl-L-tryptophan and other N-methyl amino acids, including sarcosine, which is a poor substrate. The Escherichia coli gene encoding MTOX (solA) was isolated on the basis of its sequence homology with monomeric sarcosine oxidase, a sarcosine-inducible enzyme found in many bacteria. These studies show that MTOX is expressed as a constitutive enzyme in a wild-type E. coli K-12 strain, providing the first evidence that solA is a functional gene. MTOX expression is enhanced 3-fold by growth on minimal media but not induced by N-methyl-L-tryptophan, L-tryptophan, or 3-indoleacrylate. MTOX forms an anionic flavin semiquinone and a reversible, covalent flavin-sulfite complex (K(d) = 1.7 mM), properties characteristic of flavoprotein oxidases. Rates of formation (k(on) = 5.4 x 10(-3) M(-1) s(-1)) and dissociation (k(off) = 1.3 x 10(-5) s(-1)) of the MTOX-sulfite complex are orders of magnitude slower than observed with most other flavoprotein oxidases. The pK(a) for ionization of oxidized FAD at N(3)H in MTOX (8.36) is two pH units lower than that observed for free FAD. The MTOX active site was probed by characterization of various substrate analogues that act as competitive inhibitors with respect to N-methyl-L-tryptophan. Qualitatively similar perturbations of the MTOX visible absorption spectrum are observed for complexes formed with various aromatic carboxylates, including benzoate, 3-indole-(CH(2))(n)-CO(2)(-) and 2-indole-CO(2)(-). The most stable complex with 3-indole-(CH(2))(n)-CO(2)(-) is formed with 3-indolepropionate (K(d) = 0.79 mM), a derivative with the same side chain length as N-methyl-L-tryptophan. Benzoate binding is enhanced upon protonation of a group in the enzyme-benzoate complex (pK(EL) = 6.87) but blocked by ionization of a group in the free enzyme (pK(E) = 8.41), which is attributed to N(3)H of FAD. Difference spectra observed for the aromatic carboxylate complexes are virtually mirror images of those observed with sarcosine analogues (N,N'-dimethylglycine, N-benzylglycine). Charge-transfer complexes are formed with 3-indoleacrylate, pyrrole-2-carboxylate, and CH(3)XCH(2)CO(2)(-) (X = S, Se, Te).

Anaerobiosis↗
PubMed2001

N-methyltryptophan oxidase from Escherichia coli: reaction kinetics with N-methyl amino acid and carbinolamine substrates.

N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of N-methyl-L-tryptophan (k(cat) = 4600 min(-1)). Other secondary amino acids (e.g., sarcosine) are oxidized at a slower rate. We have identified carbinolamines as a new class of alternate substrate. MTOX oxidation of the carbinolamine formed with L-tryptophan and formaldehyde yields N-formyl-L-tryptophan in a relatively slow reaction that does not compete with turnover of MTOX with N-methyl-L-tryptophan. Double reciprocal plots with N-methyl-L-tryptophan as the varied substrate are nearly parallel, but the slopes show a small, systematic variation depending on the oxygen concentration. N-Benzylglycine, a dead-end competitive inhibitor with respect to N-methyl-L-tryptophan, acts as a noncompetitive inhibitor with respect to oxygen. The results are consistent with a modified ping pong mechanism where oxygen binds to the reduced enzyme prior to dissociation of the imino acid product. MTOX is converted to a 2-electron reduced form upon anaerobic reaction with N-methyl-L-tryptophan, sarcosine, or the carbinolamine formed with L-tryptophan and formaldehyde. No evidence for a detectable intermediate was obtained by monitoring the spectral course of the latter two reactions. MTOX reduction with thioglycolate does, however, proceed via a readily detectable anionic, flavin radical intermediate. The reductive half-reaction with sarcosine at 4 degrees C exhibits saturation kinetics (k(lim) = 6.8 min(-1), K = 39 mM) and other features consistent with a mechanism in which a nearly irreversible reduction step (E(ox).S --> E(red).P) (k(lim)) is preceded by a rapidly attained equilibrium (K) between free E and the E.S complex. The 21 degrees C temperature difference can reasonably account for the 3.6-fold lower value obtained for k(lim) as compared with turnover at 25 degrees C (k(cat) = 24.5 min(-1)), suggesting that sarcosine is oxidized at a kinetically significant rate under anaerobic conditions and the reductive half-reaction is rate-limiting during turnover. These conclusions are, however, difficult to reconcile with steady-state kinetic patterns obtained with sarcosine that are consistent with a rapid equilibrium ordered mechanism with oxygen as the first substrate. The basis for the apparent stability of the MTOX.oxygen complex (K(d) = 72 microM) is unknown.

Amines↗
PubMed2001

Techno-economic evaluation of soil-aquifer treatment using primary effluent at Ahmedabad, India.

A pilot study was carried out in Sabarmati River bed at Ahmedabad, India for renovation of primary treated municipal wastewater through soil aquifer treatment (SAT) system. The infrastructure for the pilot SAT system comprised of two primary settling basins, two infiltration basins and two production wells located in the centre of infiltration basins for pumping out renovated wastewater. The performance data indicated that SAT has a very good potential for removal of organic pollutants, nutrients as well as bacteria and viruses. The SAT system was found to be more efficient and economical than the conventional wastewater treatment systems and hence recommended for adoption. A salient feature of the study is the introduction of biomat concept and its contribution in the overall treatment process.

Biomass↗
PubMed2000

Isolation and characterization of a Pseudomonas sp. strain PH1 utilizing meta-aminophenol.

Pseudomonas sp. strain PH1 was isolated from soil contaminated with pharmaceutical and dye industry waste. The isolate PH1 could use m-aminophenol as a sole source of carbon, nitrogen, and energy to support the growth. PH1 could degrade up to 0.32 mM m-aminophenol in 120 h, when provided as nitrogen source at 0.4 mM concentration with citrate (0.5 mM) as a carbon source in the growth medium. The presence of ammonium chloride as an additional nitrogen source repressed the degradation of m-aminophenol by PH1. To identify strain PH1, the 16S rDNA sequence was amplified by PCR using conserved eubacterial primers. The FASTA program was used to analyze the 16S rDNA sequence and the resulting homology patterns suggested that PH1 is a Pseudomonas.

Aminophenols↗
PubMed1999

Stress Survival of a Genetically Engineered Pseudomonas in Soil Slurries: Cytochrome P-450cam-Catalyzed Dehalogenation of Chlorinated Hydrocarbons.

Biological treatment of hazardous chemical wastes has potential as an effective, practical, and economically viable process in above the ground treatment systems that consist of both genetically engineered microorganisms (GEMs) and bioreactors with process control instruments to create ideal conditions for biodegradation. A strain of Pseudomonas putida coexpressing cytochrome P-450cam and luciferase (lux) that provides both the reductive detoxification potential of the hemoprotein and a mechanism for its reduction in the absence of "normal" P-450 redox partners was evaluated for its ability to survive and remain metabolically competent under nutrient stress in soil slurry microcosms. More than 74% of the cells of engineered Pseudomonas were culturable after 7 days of multiple nutrient (C,N,P) starvation. The diagnostic luminescence and carbon monoxide-difference spectra for the two engineered traits could be detected in a significant fraction of the surviving population. The GEM could be revived after repeated desiccation and starvation using Luria broth, benzoate, or citrate as nutrients. Soil slurries inoculated with the GEM transformed hexachloroethane (HCE) to tetrachloroethylene (tetraCE) 8-10-fold faster than uninoculated slurries. The GEM also transformed the insecticide, gamma-HCH (gamma-3,4,5,6-hexachlorocyclhexene), to gamma-3,4,5,6-tetrachlorocyclohexene (gammatetraCH) in soil slurries under subatmospheric conditions. These results indicate that GEMs can be constructed with broad substrate range detoxification catalysts such as cytochrome P-450 for remediation.

Journal Article↗
PubMed1999

Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase.

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E. coli enzyme of unknown metabolic function. Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spectrometry. The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin. The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN. Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX.

Amino Acid Sequence↗
PubMed1998

Reduction of 4-hydroxynonenal and 4-hydroxyhexenal by retinal aldose reductase.

Aldose reductase has been purified to homogeneity from bovine retina. It has an apparent molecular weight of 32,000 daltons and shares immunological and kinetic properties with the much studied aldose reductases purified from various sources. Retinal aldose reductase displays a K(m) of approximately 40 microM with 4-hydroxynonenal and 4-hydroxyhexenal, the oxidation end products of arachidonic and docosahexanoeic acids, respectively. It therefore appears that aldose reductase may constitute a major detoxification route of these toxic aldehydes in the retina.

Aldehyde Reductase↗
PubMed1997

Oxidative defense enzyme activity and mRNA levels in lenses of diabetic rats.

This study examines the mRNA expression and enzyme activity of oxidative defense enzymes during the course of streptozotocin-induced hyperglycemic cataract development. Diabetes was produced in 5-wk-old male Sprague-Dawley rats by administering streptozotocin ip and mRNA expression and enzyme activity were monitored on d 4, 8, 12, 16, 20, 40, 60, and 80; concomitantly, the onset and progress of cataract was followed by digital image analysis. Peak enzyme activity and mRNA expression were attained between d 20 and 40. Although catalase and glutathione peroxidase maintained high levels of mRNA expression through d 60, induction of CuZu-superoxide dismutase was transient, with the activity and mRNA levels returning to baseline values by d 40. There was a pronounced increase in aldose reductase activity, which gradually declined to basal levels by d 60; however, the mRNA levels remained unaltered. Other changes included a progressive loss of lenticular transparency, which declined to 40% of control by d 80. The role of antioxidant defense enzymes and, more interestingly, aldose reductase in combating oxidative stress in diabetic cataractogenesis is discussed.

Aldehyde Reductase↗
PubMed1997

Diabetes-induced apoptosis in rat kidney.

Oxidative stress has been suggested to play a crucial role in the pathogenesis of diabetic complications including nephropathy. However, the exact mechanism of diabetic nephropathy is still not clearly understood. Since oxidative stress in known to be a major component in the induction of apoptosis, we investigated the occurrence of apoptosis in diabetic rat kidney. The status of oxidative stress was determined as thiobarbituric acid reactive substances (TBARS). The TBARS in the control and diabetic rat kidney were 2.00 +/- 0.963 and 3.83 +/- 0.715 mumol/mg protein, respectively (P < 0.05). Apoptosis was determined by evaluating the DNA fragmentation using an enzyme-linked immunoassay and in situ end labeling. DNA fragmentation increased approximately fourfold in diabetic rat kidney compared to the normal kidney (P < 0.05). Apoptag in situ labeling displayed negligible apoptosis in nondiabetic kidney while significant areas of apoptosis were observed in diabetic kidney. Our results suggest that increased oxidative stress in diabetic kidney could induce apoptosis, which may contribute to the development of diabetic nephropathy.

Animals↗
PubMed1996

Semi-quantitation of mRNA by polymerase chain reaction. Levels of oxidative defense enzymes and aldose reductase in rat lenses cultured in hyperglycemic or oxidative medium.

The high sensitivity of reverse transcriptase-polymerase chain reaction for detecting low copy number mRNA transcripts has been standardized to analyze the mRNA profiles of catalase, glutathione peroxidase, CuZn-superoxide dismutase and aldose reductase, with respect to the housekeeping gene cyclophilin, in rat lenses cultured in hyperglycemic (50mM glucose) or oxidative (100 microM H2O2) media for 24, 40 and 60 hr. In response to hyperglycemia mRNA expression of catalase appeared to be inhibited at 24 hr but attained normal levels by 40 hr. On the other hand, mRNA levels of catalase were higher than normal between 40 and 60 hr in the presence of H2O2. Glutathione peroxidase mRNA abundance although enhanced in response to both hyperglycemia as well as H2O2-induced stress, displayed opposite trends with time-an increase from 24-60 hr due to hyperglycemia and a decrease to normal by 60 hr in the presence of H2O2. In contrast, CuZn-superoxide dismutase was inhibited at 50 mM glucose achieving baseline levels by 60 hr, while H2O2 elicited an induction at 24 hr which waned to basal levels by 60 hr. Interestingly, aldose reductase was unaffected by hyperglycemia but showed an appreciable increase with time upon exposure of the lens to H2O2. The role of these enzymes in cataractogenesis with regard to their respective mRNA levels is discussed.

Aldehyde Reductase↗