Mean performances, character associations and multi-environmental evaluation of chilli landraces in north western Himalayas.

Even though many varieties have been recommended across agro-climate zones of Himachal Pradesh, yet the information on stability is lacking in this State. Hence, the present investigation was carried out to identify high yielding stable genotypes among various pre-adapted landraces. The material consists of 20 chilli landraces including check i.e. DKC-8. The experiment was laid out in a RCBD. The data were recorded and analyzed to work out mean performances and the inferences were drawn for parameters of variability, correlation coefficients, path coefficients and stability analysis. As per mean performances, CS7 and CS9 were earliest in flowering, CS13 is earliest in days to ripe maturity, CS10 had highest plant height and CS9 had highest average fruit weight and ripe fruit yield plant-1. High PCV and GCV were recorded for ripe fruit yield plant-1. Heritability and genetic advance were recorded maximum for plant height in summer seasons and were recorded maximum for number of ripe fruits plant-1 in winter season. Correlation coefficients showed that number of ripe fruits plant-1 and average ripe fruit weight were positively and significantly correlated with ripe fruit yield plant-1. Path coefficient analysis in summer and winter seasons showed that average ripe fruit weight had the highest positive direct effect on ripe fruit yield plant-1. The pooled data over environments were analyzed to estimate the interaction effects between genotypes × environment. The mean sum of squares due to genotypes, environments and genotypes × environment interaction were significant for all the characteristics. CS1, CS3, CS6, CS10, CS13, CS15 were adapted to all environments, CS7 and CS9 were specifically adapted to favourable environment and CS2 was specifically adapted to unfavorable environment for 50% flowering, landraces CS1, CS2 and CS3were well adapted to all environments for ripe maturity whereas landraces CS6, CS10 and CS19 were well adapted to all environment for number of ripe fruit and ripe fruit yield.

Grain yield, adaptation and progress in breeding for early-maturing and heat-tolerant wheat lines in South Asia.

Maintaining wheat productivity under the increasing temperatures in South Asia is a challenge. We focused on developing early maturing wheat lines as an adaptive mechanism in regions suffering from terminal heat stress and those areas that require wheat adapted to shorter cycles under continual high temperature stress. We evaluated the grain yield performance of early-maturing heat-tolerant germplasm developed by CIMMYT, Mexico at diverse locations in South Asia from 2009 to 2014 and estimated the breeding progress for high-yielding and early-maturing heat-tolerant germplasm in South Asia. Each year the trial comprised of 28 new entries, one CIMMYT check (Baj) and a local check variety. Locations were classified by mega environment (ME); ME1 being the temperate irrigated locations with terminal high temperature stress, and ME5 as hot, sub-tropical, irrigated locations. Grain yield (GY), days to heading (DTH) and plant height (PH) were recorded at each location. Effect of temperature on GY was observed in both ME1 and ME5. Across years, mean minimum temperatures in ME1 and mean maximum temperatures in ME5 during grain filling had significant negative association with GY. The ME1 locations were cooler that those in ME5 in the 5 years of evaluations and had a 1-2 t/ha higher GY. A mean reduction of 20 days for DTH and 20 cm in PH was observed in ME5. Negative genetic correlations of -0.43 to -0.79 were observed between GY and DTH in South Asia during 2009-2014. Each year, we identified early-maturing germplasm with higher grain yield than the local checks. A positive trend was observed while estimating the breeding progress across five years for high-yielding early-maturing heat tolerant wheat compared to the local checks in South Asia. The results suggests the potential of the high-yielding early-maturing wheat lines developed at CIMMYT in improving wheat production and maintaining genetic gains in South Asia.

Antiarthritic activity of a standardized, multiherbal, Ayurvedic formulation containing Boswellia serrata: in vitro studies on knee cartilage from osteoarthritis patients.

A validated in vitro model of cartilage damage and published data were used showing that this model measures the chondroprotective and antiinflammatory effects of different antiarthritic drugs. In this report, this model was used to evaluate the effects of a new antiarthritic Ayurvedic formulation containing Zingiber officinale root, Tinospora cordifolia stem, Phyllanthus emblica fruit and oleoresin of Boswellia serrata. Glucosamine sulphate was used as a positive control in the study. Aqueous extracts of each drug were tested on explant cultures of knee cartilage obtained from osteoarthritis patients undergoing knee replacement surgery. The new formulation caused a sustained and statistically significant inhibition in the release of glycosaminoglycans and aggrecan by cartilage explants from these patients. This formulation also induced a transient antiinflammatory effect as measured by a reduction in the levels of nitric oxide released by explants. Furthermore, the data strongly suggest that oleoresin of B. serrata plays a crucial role in the chondroprotective and antiinflammatory activity of this formulation. In summary, this report provides the first, direct, in vitro biochemical evidence of anti-arthritic activity a new Ayurvedic formulation. This formulation significantly reduced damage of articular knee cartilage from chronic osteoarthritis patients.

Screening for disease resistance in barley cultivars against Bipolaris sorokiniana using callus culture method.

Screening for resistant barley genotypes in response to fungal toxin of Bipolaris sorokiniana was assessed on standing barley plants as well as in selected callus lines of the same. For the standing lines tested, those manifesting chlorosis in response to toxin infiltration showed a significantly slower disease progress as compared to the necrotic lines. Also, necrosis in the callus tissues of the susceptible cultivar in MS medium supplemented with different concentrations of the crude toxin was significantly higher than in the callus tissues of the chlorotic lines studied. Similar host response to the toxin in in vitro and field situations open up the possibility of screening barley cultivars for resistance to spot blotch using callus culture as against classical methods of screening in order to increase accuracy and save time and space.

Clonal variability and its relevance in generation of new pathotypes in the spot blotch pathogen, Bipolaris sorokiniana.

Spot blotch pathogen Bipolaris sorokiniana of wheat was investigated with threefold objectives: to establish a relationship between morphological and pathological variability of isolates, identify clonal genotype(s) acting as a source for the generation of new variability, and to determine the mechanism of generation of such variability in the pathogen. Isolates were collected from the leaves and seeds of field-grown wheat crop at four different sites in eastern Gangetic plains of India. Eighty-six clonal isolates derived from a single isolate (gray with white patches, Group III), which segregated in an equal proportion of parental and nonparental types, were studied. Morphological characters-i.e., colony morphology, growth rate, and sporulation-were studied along with disease-causing ability of the isolate clones. Clonal isolates were grouped into three categories. Microscopic analysis of nuclei was done to determine the causes of such variability. Morphological variability appeared to be related to the pathological variability. The isolate having epidemic potential appeared different than that acting as the reservoir for variability. The cause of such variability could be attributed either to hyphal fusion and heterokaryosis, nuclear migration and occurrence of multinucleate state, or a combination of these factors. Random Amplified Polymorphic DNA (RAPD) assay suggested that the unique fragments for different groups could be utilized as molecular markers to identify the isolates of specific groups.

Identification of molecular marker and aggressiveness for different groups of Bipolaris sorokiniana isolates causing spot blotch disease in wheat (Triticum aestivum L.).

One hundred fifty-five isolates of Bipolaris sorokiniana of wheat were studied for their morphopathological characterization. These isolates were grouped in five categories--black, brown/dull black, gray cottony growth, dull white/greenish black, and white--on the basis of their growth pattern. The frequency of the black suppressed type was maximum (45.63%), whereas the white isolate displayed lowest frequency (6.96%) in the natural population. Twenty RAPD (random amplified polymorphic DNA) primers were used to observe the variability among the identified groups of B. sorokininana. From each group, eight random isolates were investigated. A total of 143 bands were amplified, out of which 107 (74.83%) were polymorphic and 36 (25.17%) were monomorphic. On an average, the total numbers of bands generated per primer were 7.15, of which 5.35 and 1.80 were polymorphic and monomorphic, respectively. Dendrograms based on molecular polymorphism unveiled a considerable amount of diversity among the isolates. Specific DNA bands were identified for selected isolates. The distinct markers appeared to be potential enough to be employed as genetic fingerprints for future strain identification and classification. The study indicated that the RAPD primers provide an easy, rapid, and simple technique for the preliminary assessment of genetic diversity among the fungal isolates.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.].

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.

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.

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.