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K Chandran

Publications and source records attributed to K Chandran.

12 recordsLinked to original sources
PubMed2006

Observation and mathematical description of the acceleration phenomenon in batch respirograms associated with ammonium oxidation.

Two-step nitrification models are generally calibrated using short-term respirometric batch experiments. Important discrepancies appear between model predictions and experimental observations just after the pulse addition since a fast transient in the OUR profile is experimentally observed. Acceleration of the OUR appears ongoing between the substrate addition and attainment of the maximum OUR value. Among the several phenomena that could contribute to this observation, the most probable cause is the limitation of reducing equivalents required for maximal ammonia monooxygenase activity at the time of substrate addition. Ignoring acceleration would result in large parameter estimation errors from respirometric batch experiments. This work proposes a simple methodology to successfully describe (not to explain) the acceleration phenomenon estimating only two parameters. This methodology consists of introducing a Gaussian-like expression in the model.

Ammonia↗
PubMed2005

Applicability of an extant batch respirometric assay in describing dynamics of ammonia and nitrite oxidation in a nitrifying bioreactor.

Several techniques have been proposed for biokinetic estimation of nitrification. Recently, an extant respirometric assay has been presented that yields kinetic parameters for both nitrification steps with minimal physiological change to the microorganisms during the assay. Herein, the ability of biokinetic parameter estimates from the extant respirometric assay to adequately describe concurrently obtained NH4+-N and NO(2-)-N substrate depletion profiles is evaluated. Based on our results, in general, the substrate depletion profiles resulted in a higher estimate of the maximum specific growth rate coefficient, micro(max) for both NH4+-N to NO(2-)-N oxidation and NO(2-)-N to NO(3-)-N oxidation compared to estimates from the extant respirograms. The trends in the kinetic parameter estimates from the different biokinetic estimation techniques are paralleled in the nature of substrate depletion profiles obtained from best-fit parameters. Based on a visual inspection, in general, best-fit parameters from optimally designed complete respirograms provided a better description of the substrate depletion profiles than estimates from isolated respirograms. Nevertheless, the sum of the squared errors for the best-fit respirometry based parameters was outside the 95% joint confidence interval computed for the best-fit substrate depletion based parameters. Notwithstanding the difference in kinetic parameter estimates determined in this study, the different biokinetic estimation techniques still are close to estimates reported in literature. Additional parameter identifiability and sensitivity analysis of parameters from substrate depletion assays revealed high precision of parameters and high parameter correlation. Although biokinetic estimation via automated extant respirometry is far more facile than via manual substrate depletion measurements, additional sensitivity analyses are needed to test the impact of differences in the resulting parameter values on continuous reactor performance.

Ammonia↗
PubMed2001

Estimating biomass yield coefficients for autotrophic ammonia and nitrite oxidation from batch respirograms.

Kinetic characterization of biological processes via batch respirometry requires an accurate estimate of the biomass yield coefficient because it provides the stoichiometric link between biomass synthesis, substrate consumption and oxygen uptake. Expressions for biomass yield coefficients describing autotrophic ammonia and nitrite oxidation were derived from a mechanistically based electron balanced equation. We demonstrate that applying the conventional expression used to calculate the heterotrophic biomass yield results in erroneous estimates for the autotrophic biomass yield. Yield coefficients for autotrophic NH4(+)-N to NO2(-)-N oxidation and NH4(+)-N to NO3(-)-N oxidation were overestimated by 27 to 36%. Due to correlation between the maximum specific growth rate and the biomass yield, the error in yield values propagated in 30 to 40% overestimates of the maximum specific growth rate coefficient for NH4(+)-N oxidation determined from batch respirograms. Therefore, it is essential to employ the correct expression to estimate the autotrophic biomass yield coefficient from batch respirograms due its inadvertent impact on subsequent parameter estimation.

Ammonia↗
PubMed2001

Complete in vitro assembly of the reovirus outer capsid produces highly infectious particles suitable for genetic studies of the receptor-binding protein.

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins--sigma1, mu1, and sigma3--to enter host cells. sigma1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma1, mu1, and sigma3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma1. The recoated particles bound to and infected cultured cells in a sigma1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma1 protein. Additional experiments showed that recoated particles containing sigma1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma1 with respect to its structure, assembly into particles, and roles in entry.

Baculoviridae↗
PubMed2001

Peripartal cardiomyopathy: problems in diagnosis and management.

Peripartal cardiomyopathy is a rare form of heart disease in pregnancy with an unpredictable outcome. We describe one patient who presented in a decompensated state who was successfully managed with medical antifailure treatment. The etiology, management and future obstetric outcome are discussed.

Adult↗
PubMed2000

Transcriptional activities of reovirus RNA polymerase in recoated cores. Initiation and elongation are regulated by separate mechanisms.

The particle-associated reovirus polymerase synthesizes mRNA within only certain viral particle types. Reovirus cores, subviral particles lacking outer capsid proteins mu1, sigma3, and sigma1, produce mRNA and abortive transcripts. Reovirus virions, which contain complete outer capsids, cannot produce mRNA and produce few abortive transcripts. Recoated cores are virion-like particles generated by the addition of recombinant outer capsid proteins to cores. We used recoated cores to analyze transcriptional regulation by reovirus outer capsid proteins. Partially recoated particles, containing less than virion amounts of mu1 and sigma3, synthesized mRNA at levels inversely proportional to outer capsid protein levels. Fully recoated cores exhibited undetectable mRNA synthesis levels, as did virions. However, recoated cores produced high levels of abortive transcripts. Recoated core abortive transcripts remained particle-associated and appeared to inhibit further abortive transcript production. Proteolysis of recoated cores removing mu1 and sigma3 released accumulated abortive transcripts and relieved inhibition of mRNA and abortive transcript synthesis. These results suggest transcriptional elongation, but not initiation, is blocked by virion-like amounts of mu1 and sigma3. Particle-associated abortive transcripts may down-regulate transcriptional initiation. Minor outer capsid protein sigma1 had no demonstrable effect on transcriptional activities. Transcriptional regulation may ensure progeny virions do not compete with transcribing particles for ribonucleoside triphosphates.

Animals↗
PubMed2000

Applicability of two-step models in estimating nitrification kinetics from batch respirograms under different relative dynamics of ammonia and nitrite oxidation.

A mechanistically based nitrification model was formulated to facilitate determination of both NH(4)(+)-N to NO(2)(-)-N and NO(2)(-)-N to NO(3)(-)-N oxidation kinetics from a single NH(4)(+)-N to NO(3)(-)-N batch-oxidation profile by explicitly considering the kinetics of each oxidation step. The developed model incorporated a novel convention for expressing the concentrations of nitrogen species in terms of their nitrogenous oxygen demand (NOD). Stoichiometric coefficients relating nitrogen removal, oxygen uptake, and biomass synthesis were derived from an electron-balanced equation.%A parameter identifiability analysis of the developed two-step model revealed a decrease in correlation and an increase in the precision of the kinetic parameter estimates when NO(2)(-)-N oxidation kinetics became increasingly rate-limiting. These findings demonstrate that two-step models describe nitrification kinetics adequately only when NH(4)(+)-N to NO(3)(-)-N oxidation profiles contain sufficient information pertaining to both nitrification steps. Thus, the rate-determining step in overall nitrification must be identified before applying conventionally used models to describe batch nitrification respirograms.

Ammonia↗
PubMed2000

Single-step nitrification models erroneously describe batch ammonia oxidation profiles when nitrite oxidation becomes rate limiting.

Nitrification involves the sequential biological oxidation of reduced nitrogen species such as ammonium-nitrogen (NH(4)(+)-N) to nitrite-nitrogen (NO(2)(-)-N) and nitrate-nitrogen (NO(3)(-)-N). The adequacy of modeling NH(4)(+)-N to NO(3)(-)-N oxidation as one composite biochemical reaction was examined at different relative dynamics of NH(4)(+)-N to NO(2)(-)-N and NO(2)(-)-N to NO(3)(-)-N oxidation. NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation by a mixed nitrifying consortium were uncoupled using selective inhibitors allylthiourea and sodium azide. The kinetic parameters of NH(4)(+)-N to NO(2)(-)-N oxidation (q(max,ns) and K(S,ns)) and NO(2)(-)-N to NO(3)(-)-N oxidation (q(max,nb) and K(S,nb)) were determined by a rapid extant respirometric technique. The stoichiometric coefficients relating nitrogen removal, oxygen uptake and biomass synthesis were derived from an electron balanced equation. NH(4)(+)-N to NO(2)(-)-N oxidation was not affected by NO(2)(-)-N concentrations up to 100 mg NO(2)(-)-N L(-1). NO(2)(-)-N to NO(3)(-)-N oxidation was noncompetitively inhibited by NH(4)(+)-N but was not inhibited by NO(3)(-)-N concentrations up to 250 mg NO(3)(-)-N L(-1). When NH(4)(+)-N to NO(2)(-)-N oxidation was the sole rate-limiting step, complete NH(4)(+)-N to NO(3)(-)-N oxidation was adequately modeled as one composite process. However, when NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation were both rate limiting, the estimated lumped kinetic parameter estimates describing NH(4)(+)-N to NO(3)(-)-N oxidation were unrealistically high and correlated. These findings indicate that the use of single-step models to describe batch NH(4)(+) oxidation yields erroneous kinetic parameters when NH(4)(+)-to-NO(2)(-) oxidation is not the sole rate-limiting process throughout the assay. Under such circumstances, it is necessary to quantify NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation, independently.

Ammonia↗
PubMed1999

In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3.

Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.

Animals↗
PubMed1998

Protease cleavage of reovirus capsid protein mu1/mu1C is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle.

Mammalian reovirus virions undergo partial disassembly of the outer capsid upon exposure to proteases in vitro, producing infectious subvirion particles (ISVPs) that lack protein sigma3 and contain protein mu1/mu1C as endoprotease-generated fragments mu1delta/delta and phi. ISVPs are thought to be required for two early steps in reovirus infection: membrane penetration and activation of the particle-bound viral transcriptase complexes. Genetic and biochemical evidence implicates outer-capsid protein mu1 in both these steps. To determine whether the cleavage of mu1/mu1C is relevant to the unique properties of ISVPs, we analyzed the properties of novel subvirion particles that lacked sigma3 yet retained mu1/mu1C in an uncleaved but cleavable form. These detergent-plus-protease subvirion particles (dpSVPs) were produced by treating virions with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents. Infections with dpSVPs in murine L or canine MDCK cells provided evidence that the cleavage of mu1/mu1C during viral entry into these cells is dispensable for reovirus infection. Additionally, dpSVPs behaved like ISVPs in their capacity to permeabilize lipid bilayers and to undergo transcriptase activation in vitro, supporting the conclusion that cleavage of mu1/mu1C to mu1delta/delta and phi during viral entry is not required for either membrane penetration or transcriptase activation in cells. The capacity of alkyl sulfate detergents to inhibit the cleavage of mu1/mu1C in a reversible fashion suggests a specific association between virus particle and detergent micelles that may mimic virus particle-phospholipid membrane interactions during reovirus entry into cells.

Animals↗
PubMed1996

Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls.

Hepatic uroporphyria is a well-known effect of halo- genated aromatic hydrocarbons in mammalian and avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB; IUPAC 77) when the heme precursor delta-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (approximately 80%) and heptacarboxylporphyrin (approximately 20%). Uroporphyria did not occur in cells treated with TCDD or 3,3',4,4'-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4',5-tetrachlorobiphenyl (IUPAC 81) and 3,3',4,4',5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3',4,4'-pentachlorobiphenyl (IUPAC 105) or 2,3',4,4'5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin 0-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. alpha-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this prophyria. Addition of 3,3',4,4'-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model System for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.

Animals↗