Microsatellite markers for resistance to spot blotch in spring wheat.
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Crosses, Genetic↗
Biomedical subjects
A K Joshi
Publications and source records attributed to A K Joshi.
Production of L-phenylacetyl carbinol by immobilized cells of Saccharomyces cerevisiae.
Conversion of benzaldehyde to L-phenylacetyl carbinol (L-PAC) was achieved with immobilized, growing cells of Saccharomyces cerevisiae in different reactors. Product formation increased (31%) with the subsequent initial reuses of the entrapped cells. Biomass production and PAC formation depleted (40 and 57%, respectively) after 4-5 continuous growth and biotransformation cycles. With the regeneration of the biocatalysts, catalytic activity of the cells was resumed. The highest yields were in a stirred tank reactor (29 g PAC) from 77 g benzeldehyde with 14 repeated uses of entrapped cells.
Acetone↗
Studies on physiology, zoospore morphology and entomopathogenic potential of the aquatic oomycete: Lagenidium giganteum.
The oomycete Lagenidium giganteum, a facultative parasite of mosquito larvae requires exogenous sterols for the genesis of zoospores when grown saprobically. Growth media prepared from oil rich materials such as soy or sunflower seed were very effective inducers of virulent zoospores. The external morphology of zoospores of L. giganteum was studied with the aid of philips scanning electron microscope 515. Zoospores were ovoid, bluntly pointed with the groove parallel to the long axis and 0.7 x 1.4 microm. Insect cell walls are known to contain lipid and chitin. L. giganteum was tested for chitinase activity and found to possess 0.76 +/- SD0.14 chitinase activity. Use of oil seed for growth of the organism confirms phospholipase activity. Phospholipase production was studied further by egg-yolk plate method. Presence of these two key enzymes that can initiate host cell damage suggests the entomopathogenic potential of L. giganteum. L. giganteum failed to grow at 37 degrees C limiting its effectiveness in warmer climates. Introduction of this organism to variety of habitats with various mosquito species will demonstrate the efficacy of the organism as a bioinsecticide.
Animals↗
Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro.
An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.
Alcohol Oxidoreductases↗
Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.
beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit. Reevaluation of the side-by-side, antiparallel subunit model.
The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.
Acetone↗
Double-blind comparison of carbetocin versus oxytocin in prevention of uterine atony after cesarean section.
OBJECTIVE: The goal of this study was to compare carbetocin, a long-acting oxytocin analog, with oxytocin in the prevention of uterine atony after cesarean section. STUDY DESIGN: We enrolled 694 patients undergoing elective cesarean section in a Canadian multicenter, double-blind, randomized clinical trial. We compared the effect of a single 100 microg dose of carbetocin with that of a standard 8-hour infusion of oxytocin. The primary outcome was the proportion of patients requiring additional oxytocic intervention for uterine atony. A variable sample size, sequential design was used. RESULTS: The overall oxytocic intervention rate was 7.4%. The odds of treatment failure requiring oxytocic intervention was 2.03 (95% confidence interval 1.1 to 2.8) times higher in the oxytocin group compared with the carbetocin group, respectively, 32 of 318 (10.1%) versus 15 of 317 (4.7%), P <.05. CONCLUSIONS: Carbetocin, a new drug for the prevention of uterine atony, appears to be more effective than a continuous infusion of oxytocin and has a similar safety profile.
Adult↗
Fatty acid synthase dimers containing catalytically active beta-ketoacyl synthase or malonyl/acetyltransferase domains in only one subunit can support fatty acid synthesis at the acyl carrier protein domains of both subunits.
A double-tagging, dual affinity chromatographic procedure, which permits isolation of dimers independently mutated in each subunit, has been exploited to probe the functional topology of the animal fatty acid synthase. Dimers were engineered in which the chain-terminating thioesterase reaction was compromised by mutation of the (active-site) serine residue in both subunits; these dimers assembled two long-chain fatty acyl moieties, which remained covalently linked to the 4'-phosphopantetheine residues of the two acyl carrier protein domains. Significantly, dimers that contained an additional mutation that compromised the activity of either the beta-ketoacyl synthase or malonyl/acetyltransferase activity in only one subunit also assembled two long-chain acyl moieties. In contrast, in a control experiment, introduction of an additional mutation that compromised the function of the acyl carrier protein domain in only one subunit resulted in the assembly of only one long-chain acyl moiety per dimer. Because the beta-ketoacyl synthase and malonyl/acetyltransferase domains are located near the amino terminus of the polypeptide and the acyl carrier protein domain near the carboxyl terminus, these results support a modified model for the animal fatty acid synthase in which head-to-tail functional contacts are possible both within as well as between subunits.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Differential affinity labeling of the two subunits of the homodimeric animal fatty acid synthase allows isolation of heterodimers consisting of subunits that have been independently modified.
To explore the domain interactions that are required for catalytic activity of the multifunctional, homodimeric fatty acid synthase (FAS), we have formulated a strategy that allows isolation of modified dimers containing independently mutated subunits. Either a hexahistidine or a FLAG octapeptide tag was incorporated into the FAS at either the amino terminus, within an internal noncatalytic domain, or at the carboxyl terminus. The presence of the tags had no effect on the activity of the wild-type FAS. His-tagged dimers were mixed with FLAG-tagged dimers, and the subunits were randomized to produce a mixture of His-tagged homodimers, FLAG-tagged homodimers, and doubly tagged heterodimers. The doubly tagged heterodimers could be purified to homogeneity by chromatography on an anti-FLAG immunoaffinity column followed by a metal ion chelating column. This procedure for isolation of FAS heterodimers was utilized to determine whether the two centers for fatty acid synthesis in the FAS dimer can function independently of each other. Doubly tagged heterodimers, consisting of one wild-type subunit and one subunit in which the thioesterase activity had been eliminated, either by mutation or by treatment with phenylmethanesulfonyl fluoride, have 50% of the wild-type thioesterase activity and, in the presence of substrates, accumulate a long chain fatty acyl moiety on the modified subunit, thus blocking further substrate turnover at this center. Nevertheless, the ability of the heterodimer to synthesize fatty acids is also 50% of the wild-type FAS, demonstrating that an individual center for fatty acid synthesis has the same activity when paired with either a functional or nonfunctional catalytic center.
Affinity Labels↗
The malonyl/acetyltransferase and beta-ketoacyl synthase domains of the animal fatty acid synthase can cooperate with the acyl carrier protein domain of either subunit.
The active form of the animal fatty acid synthase (FAS) is a dimer of identical multifunctional polypeptides, each containing seven discrete functional domains, that cooperate to form two centers for palmitate synthesis. To assess the importance of domain cooperation across the subunit interface in the reaction mechanism, we have utilized a strategy based on complementation analysis in vitro of modified FASs carrying critical mutations in specific catalytic domains. Homodimeric FASs carrying the same mutation(s) in both subunits are unable to synthesize fatty acids. As predicted by the current head-to-tail model for the animal FAS, heterodimeric FASs formed between the acyl carrier protein (ACP) mutant and either the beta-ketoacyl synthase (KS) or malonyl/acetyltransferase (MAT) are active in palmitate synthesis, confirming that the KS and MAT domains can cooperate with the ACP domain of the opposite subunit. Contrary to this model however, heterodimeric FASs formed between the KS and MAT mutants, between a MAT, ACP double mutant, and a KS mutant, and between a KS, ACP double mutant, and a MAT mutant are also active in palmitate synthesis, indicating that the MAT and KS domains can also cooperate with the ACP domain of the same subunit. The results of this study reveal an unanticipated element of redundancy in the FAS reaction mechanism in that the amino-terminal KS and MAT domains can make functional contact with the penultimate carboxy-terminal ACP domain of either subunit. A revised model for the FAS is proposed in which the substrate loading and condensation reactions can be catalyzed either by one of the two subunits or by cooperation between domains across the subunit interface.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Production of a polyhydroxyalkanoate biopolymer in insect cells with a modified eucaryotic Fatty Acid synthase.
[This corrects the article on p. 2540 in vol. 62, PMID: 8779593.].
Journal Article↗
Characterization of the interthiol acyltransferase reaction catalyzed by the beta-ketoacyl synthase domain of the animal fatty acid synthase.
The enzyme activity responsible for translocation of saturated acyl chains from the 4'-phosphopantetheine of the acyl carrier protein to the active site cysteine of the beta-ketoacyl synthase in the animal fatty acid synthase has been identified. An enzyme assay was devised that allows uncoupling of the interthiol transfer step from the condensation reaction. Experiments with various fatty acid synthase mutants indicate clearly that catalysis of the transfer of saturated acyl moieties from the 4'-phosphopantetheine thiol to the active site cysteine thiol, Cys-161, is an inherent property of the beta-ketoacyl synthase domain. Catalytic efficiency of the interthiol transferase increases from C2 to C12 and decreases with increasing chain-lengths beyond C12. Malonyl, beta-hydroxybutyryl, and crotonyl thioesters are not substrates for the transferase, whereas the beta-ketobutyryl moiety is a poor substrate. These features of the substrate specificity are exactly as predicted for a transferase that fulfills the proposed role in the fatty acid synthase reaction sequence and indicate that this activity plays an important role in determining the overall specificity of the beta-ketoacyl synthase reaction.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Mapping of functional interactions between domains of the animal fatty acid synthase by mutant complementation in vitro.
Polypeptides of the animal fatty acid synthase (FAS) consist of three amino-terminal catalytic domains, beta-ketoacyl synthase, malonyl/acetyltransacylase, and dehydrase, separated by a 600-residue structural core from four carboxyl-terminal catalytic domains, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase. In the active dimeric form of the protein the two identical multifunctional polypeptides are oriented head-to-tail such that two sites for palmitate synthesis are formed at the subunit interface. In order to map the functional interactions between domains of the two subunits that contribute to the two sites of synthesis, we have utilized a strategy based on complementation analysis in vitro of modified FASs carrying mutations in specific catalytic domains. Homodimeric mutant FASs lacking functional beta-ketoacyl synthase (KS-), dehydrase (DH-), acyl carrier protein (ACP-), or thioesterase (TE-) domains, as well as heterodimers formed between ACP- and TE- subunits, between ACP- and DH- subunits, and between DH- and TE- subunits, were unable to synthesize fatty acids. However, heterodimers formed between KS- and either DH-, ACP-, or TE- subunits regained partial FAS activity. These data indicate that the dehydrase domain, although located in the amino-terminal half of the polypeptide, should be assigned to the complementation group located in the carboxy-terminal half that includes the acyl carrier protein and thioesterase domains. Thus, the current model for the animal FAS must be revised to reflect the finding that the two constituent polypeptides are not simply positioned side-by-side in a fully extended conformation but are coiled in a manner that allows the dehydrase domain to access the beta-hydroxyacyl-ACP located more than 1100 residues distant on the same subunit.
Animals↗
Production of L-phenylacetylcarbinol by free and immobilized yeast cells.
Production of L-phenylacetylcarbinol (L-PAC) through biotransformation of benzaldehyde by free and immobilized cells of the yeast Saccharomyces cerevisiae has been attempted. L-PAC production was found to be maximum (0.4 microliter/ml) when anaerobically grown free cells were used as biocatalyst during aerobic biotransformation for two hours with magnetically stirred bioreactor. Growth under oxygen limited conditions led to accumulation of higher amount of pyruvate decarboxylase enzyme and co-substrate, pyruvate, resulting in higher L-PAC formation. L-PAC yield was low when biotransformations were carried out anaerobically either for aerobically or anaerobically grown free cells. Free cells were found to be more efficient biocatalyst for L-PAC production, as compared with the immobilized cells, with the investigated benzaldehyde concentration (0.3% v/v) and cell density (17.5% w/v). The study has explored and indicated the possibility of optimizing the yield of L-PAC by growing the yeast cells under oxygen limited condition for suitable aerobic mode of benzaldehyde biotransformation.
Acetone↗
Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases.
The histidine-binding protein, HisJ, is the soluble receptor for the periplasmic histidine permease of Salmonella typhimurium. The receptor binds the substrate in the periplasm, interacts with the membrane-bound complex, transmits a transmembrane signal to hydrolyze ATP, and releases the ligand for translocation. HisJ, like other periplasmic receptors, has two lobes that are apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation), burying deeply the ligand. Such receptors are postulated to interact with the membrane-bound complex with high affinity in their liganded conformation, and, upon substrate translocation, to undergo a reduction in affinity and therefore be released. Here we show that in contrast to the current postulate, liganded and unliganded receptors have equal affinity for the membrane-bound complex. The affinity is measured both by chemical cross-linking and co-sedimentation procedures. An ATPase activity assay is also used to demonstrate the interaction of unliganded receptor with the membrane-bound complex. These findings support a new model for the transport mechanism, in which the soluble receptor functions independently of the commonly accepted high-low affinity switch.
ATP-Binding Cassette Transporters↗
Maternal asthma and idiopathic preterm labor.
Previous studies suggest that women with asthma are at increased risk of preterm birth. Moreover, drugs (especially beta-agonists) used to treat asthma are also used to treat preterm labor. The authors carried out a case-control study of 555 women from three hospital centers with idiopathic preterm labor (< 37 weeks), including two overlapping (i.e., non-mutually exclusive) subsamples: cases with early idiopathic preterm labor (< 34 weeks) and cases with idiopathic recurrent preterm labor (< 37 weeks plus a previous history of preterm delivery or second-trimester miscarriage). Controls were matched to cases according to race and smoking history prior to and during pregnancy. All subjects responded in person to questions about atopic, respiratory, obstetric, and sociodemographic histories. Subjects in the early and recurrent preterm labor subsamples were also asked to undergo spirometric testing with methacholine challenge 6-12 weeks after delivery. Cases were significantly more likely to report histories of asthma symptoms and physician-diagnosed asthma (matched odds ratios of 2-3) than controls, particularly those cases with recurrent preterm labor. No significant associations were observed, however, with methacholine responsiveness. These results could not be explained by residual confounding by smoking or other variables, nor by selective recall of asthma symptoms and histories by cases. Women with asthma are at increased risk of idiopathic preterm labor. The fact that no such association was seen with methacholine responsiveness suggests that nonatopic, noncholinergic mechanisms may link bronchial and uterine smooth muscle lability.
Adult↗
Physicians' beliefs and behaviour during a randomized controlled trial of episiotomy: consequences for women in their care.
OBJECTIVE: To evaluate whether physicians' beliefs concerning episiotomy are related to their use of procedures and to differential outcomes in childbirth. DESIGN: Post-hoc cohort analysis of physicians and patients involved in a randomized controlled trial of episiotomy. SETTING: Two tertiary care hospitals and one community hospital in Montreal. PARTICIPANTS: Of the 703 women at low risk of medical or obstetric problems enrolled in the trial we studied 447 women (226 primiparous and 221 multiparous) attended by 43 physicians. Subjects attended by residents or nurses were excluded. MAIN OUTCOME MEASURES PATIENTS: intact perineum v. perineal trauma, length of labour, procedures used (instrumental delivery, oxytocin augmentation of labour, cesarean section and episiotomy), position for birth, rate of and reasons for not assigning women to a study arm, postpartum perineal pain and satisfaction with the birth experience, physicians: beliefs concerning episiotomy. RESULTS: Women attended by physicians who viewed episiotomy very unfavorably were more likely than women attended by the other physicians to have an intact perineum (23% v. 11% to 13%, p < 0.05) and to experience less perineal trauma. The first stage of labour was 2.3 to 3.5 hours shorter for women attended by physicians who viewed episiotomy favourably than for women attended by physicians who viewed episiotomy very unfavorably (p < 0.05 to < 0.01), and the former physicians were more likely to use oxytocin augmentation of labour. Physicians who viewed episiotomy more favourably failed more often than those who viewed the procedure very unfavourably to assign patients to a study arm late in labour (odds ratio [OR] 1.88, p < 0.05), both overall and because they felt that "fetal distress" or cesarean section necessitated exclusion of the subject. They used the lithotomy position for birth more often (OR 3.94 to 4.55, p < 0.001), had difficulty limiting episiotomy in the restricted-use arm of the trial and diagnosed fetal distress and perineal inadequacy more often than the comparison groups. The patients of physicians who viewed episiotomy very favourably experienced more perineal pain (p < 0.01), and of those who viewed episiotomy favourably and very favourably experienced less satisfaction with the birth experience (p < 0.01) than the patients of physicians who viewed the procedure very unfavourably. CONCLUSIONS: Physicians with favourably views of episiotomy were more likely to use techniques to expedite labour, and their patients were more likely to have perineal trauma and to be less satisfied with the birth experience. This evidence that physician beliefs can influence patient outcomes has both clinical and research implications.
Adult↗
Structure/function analysis of the periplasmic histidine-binding protein. Mutations decreasing ligand binding alter the properties of the conformational change and of the closed form.
The periplasmic histidine-binding protein, HisJ, is a receptor for the histidine permease of Salmonella typhimurium. Receptors of this type are composed of two lobes that are far apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation). The binding of the ligand, in a cleft between the lobes, stabilizes the closed conformation. Such receptors have several functions in transport: interaction with the membrane-bound complex, transmission of a transmembrane signal to hydrolyze ATP, and receiving a signal to open the lobes and release the ligand. In this study the mechanism of action of HisJ was further investigated using mutant proteins defective in ligand binding activity and closed form-specific monoclonal antibodies (Wolf, A., Shaw, E. W., Nikaido, K., and Ames G. F.-L. (1994) J. Biol. Chem. 269, 23051-23058). Y14H is defective in stabilization of the closed form, does not assume the closed empty form, and assumes an altered closed liganded form. T121A and G119R are similar to Y14H, but assume a normal closed liganded form. S72P binds the ligand to the open form, but does not assume a recognizable closed form. S92F is defective in the ability to undergo conformational change and to stabilize the closed form. All other mutant proteins appear to fall within one of these four categories. The biochemical characterization of these mutant proteins agrees with the structural analysis of the protein. We suggest that mutant proteins that do not assume the normal closed form, in addition to their defect in ligand binding, fail to interact with the membrane-bound complex and/or to transmit transmembrane signals.
Antibodies, Bacterial↗