Utilization of crop wild relatives for biotic and abiotic stress management in Indian mustard [Brassica juncea (L.) Czern. & Coss.].

Brassica juncea (L.) Czern. & Coss. (Indian mustard) is an economically important edible oil crop. Over the years, plant breeders have developed many elite varieties of B. juncea with better yield traits, but research work on the introgression of stress resilience traits has largely been lagging due to scarcity of resistant donors. Crop wild relatives (CWRs) are the weedy relatives of domesticated plant species which are left unutilized in their natural habitat due to the presence of certain undesirable alleles which hamper their yield potential, and thus, their further domestication. CWRs of B. juncea namely include Sinapis alba L. (White mustard), B. tournefortii Gouan. (African mustard), B. fruticulosa Cirillo (Twiggy turnip), Camelina sativa L. (Gold-of-pleasure), Diplotaxis tenuisiliqua Delile (Wall rocket), D. erucoides L. (White wall rocket), D. muralis L. (Annual wall rocket), Crambe abyssinica R.E.Fr. (Abyssinian mustard), Erucastrum gallicum Willd. (Common dogmustard), E. cardaminoides Webb ex Christ (Dogmustard), Capsella bursa-pastoris L. (Shepherds purse), Lepidium sativum L. (Garden Cress) etc. These CWRs have withstood several regimes of biotic and abiotic stresses over the past thousands of years which led them to accumulate many useful alleles contributing in resistance against various environmental stresses. Thus, CWRs could serve as resourceful gene pools for introgression of stress resilience traits into Indian mustard. This review summarizes research work on the introgression of resistance against Sclerotinia stem rot (caused by Sclerotinia sclerotiorum), Alternaria blight (caused by Alternaria brassicae), white rust (caused by Albugo candida), aphid attack, drought and high temperature from CWRs into B. juncea. However, various pre- and post-fertilization barriers due to different ploidy levels are major stumbling blocks in the success of such programmes, therefore, we also insightfully discuss how the advances made in -omics technology could be helpful in assisting various breeding programmes aiming at improvisation of stress resilience traits in B. juncea.

Structural analysis of LpqY, a substrate-binding protein from the SugABC transporter of Mycobacterium tuberculosis, provides insights into its trehalose specificity.

The LpqY-SugABC transporter of Mycobacterium tuberculosis (Mtb) salvages residual trehalose across the cell membrane, which is otherwise lost during the formation of cell-wall glycoconjugates in the periplasm. LpqY, a substrate-binding protein from the SugABC transporter, acts as the primary receptor for the recognition of trehalose, leading to its transport across the cell membrane. Since trehalose is crucial for the survival and virulence of Mtb, trehalose receptors should serve as important targets for novel drug design against tuberculosis. In order to comprehend the detailed architecture and substrate specificity, the first crystal structures of both apo and trehalose-bound forms of M. tuberculosis LpqY (Mtb-LpqY) are presented here at 2.2 and 1.9 Šresolution, respectively. The structure exhibits an N-lobe and C-lobe and is predominantly composed of a globular α/ß domain connected by a flexible hinge region concealing a deep binding cleft. Although the trehalose-bound form of Mtb-LpqY revealed an open ligand-bound conformation, the glucose moieties of trehalose are seen to be strongly held in place by direct and water-mediated hydrogen bonds within the binding cavity, producing a Kd of 6.58 ± 1.21 µM. These interactions produce a distinct effect on the stereoselectivity for the α-1,1-glycosidic linkage of trehalose. Consistent with the crystal structure, molecular-dynamics simulations further validated Asp43, Asp97 and Asn151 as key residues responsible for strong and stable interactions throughout a 1 µs time frame, thus capturing trehalose in the binding cavity. Collectively, the results provide detailed insights into how the structure and dynamics of Mtb-LpqY enable it to specifically bind trehalose in a relaxed conformation state.

PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone.

Diagnostic value of the total antioxidant capacity (TAC) in human seminal plasma.

OBJECTIVE: To establish cutoff value, sensitivity, specificity and intra- and interobserver variability of total antioxidant capacity (TAC) in seminal plasma from healthy donors (controls) and infertile patients. DESIGN: Seminal plasma from proven fertile donors (n = 55), nonproven fertile donors (n = 45), and infertile patients (n = 42) were examined for TAC level. SETTING: Reproductive research center in a tertiary care hospital. PATIENT(S): Infertile patients from male infertility clinic of various diagnoses. INTERVENTION(S): Seminal plasma TAC measurement by a colorimetric assay using the TAC assay kit, receiver operating characteristics curve. MAIN OUTCOME MEASURE(S): Seminal plasma TAC levels, cutoff value, sensitivity, and specificity. RESULT(S): Proven fertile donors showed higher TAC values (median and range): 1700 (1440-2290 microM); compared with the infertile patients: 1310 (1040-1600 microM). The best cutoff to distinguish between fertile controls and infertile men was 1420 microM. At this threshold, specificity was 64% and sensitivity 76%. CONCLUSION(S): Total antioxidant capacity of the seminal plasma as measured by the colorimetric assay is a reliable and simple test for the diagnosis and management of male infertility.

Regionalized calcium signaling in zebrafish fertilization.

Fertilization involves an initial, highly localized signal delivered by the sperm, which becomes amplified by a signal transduction cascade to impact the entire oocyte cytoplasm. The zebrafish oocyte presents a unique opportunity to study this process since fertilization always occurs at the micropyle, allowing the investigator to image the earliest steps in the oocyte activation process. The objective of the present study was to characterize the amplification of the sperm-induced calcium transient in the zebrafish oocyte and test the role of Fyn kinase in this process. Confocal fluorescence microscopy revealed that the sperm-induced calcium transient was composed of two elements, one of which was unique to the oocyte cortex and a second, slower transient that occurred in the central cytoplasm of the oocyte. The cortical transient was initiated immediately deep to the micropyle, became amplified at the animal pole, and progressed peripherally through the oocyte cortex. This was followed by a slower transient that occurred in the central cytoplasm of the oocyte. Several lines of evidence indicate that calcium release in these two compartments may be regulated differently. The calcium transient in the oocyte cortex is highly sensitive to inhibition by Fyn-SH2 domain containing fusion proteins, while the central cytoplasmic transient is relatively resistant to this treatment. Oocytes stimulated by injection of a soluble extract prepared from zebrafish sperm respond only with a cortical calcium transient initiated at the micropyle, while oocytes stimulated parthenogenetically by hypotonic shock exhibit a defective cortical transient but a normal transient in the central cytoplasm. Analysis of the subcellular distribution of Fyn kinase and the IP3 receptor reveal that these important signaling components are highly enriched in the oocyte cortex, a factor which may facilitate a faster propagation of the calcium transient in this compartment. In summary, analysis of calcium signaling in the zebrafish oocyte requires attention to morphologically distinct compartments of the oocyte and it is likely that these compartments are controlled by different biochemical events.

Fertilization triggers localized activation of Src-family protein kinases in the zebrafish egg.

Fertilization triggers activation of Src-family kinases in eggs of various species including marine invertebrates and lower vertebrates. While immunofluorescence studies have localized Src-family kinases to the plasma membrane or cortical cytoplasm, no information is available regarding the extent to which these kinases are activated in different regions of the zygote. The objective of the present study was to detect the subcellular distribution of activated Src-family kinases in the fertilized zebrafish egg. An antibody specific for the active, non-phosphorylated form of Src-family PTKs was used to detect these activated kinases by immunofluorescence. The results demonstrate that Fyn, and possibly other Src family members are activated by dephosphorylation of the C-terminal tyrosine at fertilization. The activated Src-family kinases are asymmetrically distributed around the egg cortex with an area of higher kinase activity localized adjacent to the micropyle near the presumptive animal pole. Fertilization initially caused elevation of kinase activity in the cytoplasm underlying the micropyle, but this quickly spread to involve the entire zygote cortex. Later, during egg activation, formation of the blastodisc involved concentration of active Src-family kinase in the blastodisc cortex. As cytokinesis began, activated Src-family kinases were no longer limited to the cortex, but became more evenly distributed in the clear apical cytoplasm of the blastomeres. The results demonstrate that the cortex of the zebrafish egg is functionally differentiated and that fertilization triggers localized activation of Src-family kinases at the point of sperm entry, which subsequently progresses through the entire egg cortex.

Differential expression of small-conductance Ca2+-activated K+ channels SK1, SK2, and SK3 in mouse atrial and ventricular myocytes.

Small-conductance Ca2+-activated K+ channels (SK channels, KCa channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca2+ with membrane potential. We recently reported for the first time the functional existence of SK2 (KCa2.2) channels in human and mouse cardiac myocytes. Here, we report cloning of SK1 (KCa2.1) and SK3 (KCa2.3) channels from mouse atria and ventricles using RT-PCR. Full-length transcripts and their variants were detected for both SK1 and SK3 channels. Variants of mouse SK1 channel (mSK1) differ mainly in the COOH-terminal structure, affecting a portion of the sixth transmembrane segment (S6) and the calmodulin binding domain (CaMBD). Mouse SK3 channel (mSK3) differs not only in the number of polyglutamine repeats in the NH2 terminus but also in the intervening sequences between the polyglutamine repeats. Full-length cardiac mSK1 and mSK3 show 99 and 91% nucleotide identity with those of mouse colon SK1 and SK3, respectively. Quantification of SK1, SK2, and SK3 transcripts between atria and ventricles was performed using real-time quantitative RT-PCR from single, isolated cardiomyocytes. SK1 transcript was found to be more abundant in atria compared with ventricles, similar to the previously reported finding for SK2 channel. In contrast, SK3 showed similar levels of expression in atria and ventricles. Together, our data are the first to indicate the presence of the three different isoforms of SK channels in heart and the differential expression of SK1 and SK2 in mouse atria and ventricles. Because of the marked differential expression of SK channel isoforms in heart, specific ligands for Ca2+-activated K+ currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes.

Role of Fyn kinase in signaling associated with epiboly during zebrafish development.

The function of Fyn kinase during zebrafish development through the blastula stage was investigated through the use of dominant-negative constructs designed to suppress the function of zebrafish c-Fyn. Microinjection of SH2 domain-containing fusion protein or mRNA encoding the mutated, catalytically inactive Fyn at 45 min post-insemination had no significant effect during cleavage and did not inhibit formation of the yolk syncitial layer. Smoothing of the enveloping cell layer at the midblastula transition occurred normally and expression of bon/mixer and mezzo, zygotic transcription factors indicated that activation of the zygotic genome did occur. Signaling pathways involved with axis determination such as beta-catenin, activin, and nodal appeared to function normally as evidenced by expression of boz, goosecoid, and mezzo. However, while formation of the yolk syncitial layer was normal, the marginal blastomeres failed to migrate toward the vegetal pole and epiboly did not occur, a phenotype similar but distinct from that resulting from suppression of c-Yes kinase. The block to development was prevented by co-injection of c-Fyn mRNA with the dominant-negative construct indicating that it was a specific effect. Injection of the dominant-negative mRNA into individual blastomeres indicated that the effect was exerted on the intrinsic ability of the individual blastomeres to respond to signals directing epiboly and not on the signals themselves. Analysis of the pattern of calcium signaling in experimental and control embryos demonstrated that the elevated [Ca2+]i characteristic of the marginal blastomeres was suppressed. Together, these observations indicate that Fyn kinase plays an important role in epiboly, possibly through its effects in calcium signaling.

The effects of intracellular Ca2+ on cardiac K+ channel expression and activity: novel insights from genetically altered mice.

We tested the hypothesis that chronic changes in intracellular Ca(2+) (Ca(2+)(i)) can result in changes in ion channel expression; this represents a novel mechanism of crosstalk between changes in Ca(2+) cycling proteins and the cardiac action potential (AP) profile. We used a transgenic mouse with cardiac-specific overexpression of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) isoform 1a (SERCA1a OE) with a significant alteration of SERCA protein levels without cardiac hypertrophy or failure. Here, we report significant changes in the expression of a transient outward K(+) current (I(to,f)), a slowly inactivating K(+) current (I(K,slow)) and the steady state current (I(SS)) in the transgenic mice with resultant prolongation in cardiac action potential duration (APD) compared with the wild-type littermates. In addition, there was a significant prolongation of the QT interval on surface electrocardiograms in SERCA1a OE mice. The electrophysiological changes, which correlated with changes in Ca(2+)(i), were further corroborated by measuring the levels of ion channel protein expression. To recapitulate the in vivo experiments, the effects of changes in Ca(2+)(i) on ion channel expression were further tested in cultured adult and neonatal mouse cardiac myocytes. We conclude that a primary defect in Ca(2+) handling proteins without cardiac hypertrophy or failure may produce profound changes in K(+) channel expression and activity as well as cardiac AP.

Role of Yes kinase during early zebrafish development.

We have identified the Yes kinase in zebrafish eggs and investigated its role in development of the zebrafish embryo. In situ hybridization as well as immunofluorescence techniques demonstrated that Yes kinase is maternally expressed and is localized to the cortical region of the unfertilized egg. Fertilization resulted in concentration of Yes kinase to the blastodisc where it continued to be localized to the blastoderm cells through cleavage, gastrulation, and later development. Yes kinase activity was found to decrease abruptly at fertilization, then increase progressively during epiboly, and was maintained at high levels throughout gastrulation. The role of Yes kinase in development was tested by treating embryos with chemical protein tyrosine kinase (PTK) inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) and by injection of antisense morpholinos. Both treatments resulted in the arrest of development at the beginning of the epiboly. Co-immunoprecipitation studies demonstrated that Yes kinase participates in a stable complex with focal adhesion kinase (FAK), which is phosphorylated in vitro. These results demonstrate that Yes kinase plays an important role in epiboly and indicate that Yes kinase participates in signaling by focal adhesion kinase during early development.

Characterization of a KCNQ1/KVLQT1 polymorphism in Asian families with LQT2: implications for genetic testing.

Congenital long QT syndrome (LQTS) is a genetic disease that predisposes affected individuals to arrhythmias, syncope, and sudden death. Mutations in several ion channel genes have been discovered in different families with LQTS: KCNQ1 (KVLQT1, LQT1), KCNH2 (HERG, LQT2), SCN5A (LQT3), KCNE1 (minK, LQT5), and KCNE2 (MiRP1, LQT6). Previously, the P448R-KVLQT1 missense mutation has been reported as an LQT1-causing mutation. In this report, we demonstrate the presence of the P448R polymorphism in two, unrelated Chinese LQTS families. Although absent from 500 reference alleles derived from 150 white and 100 African-American subjects, P448R was present in 14% of healthy Chinese volunteers. Given the inconsistencies between the genotype (LQT1) and clinical phenotype (LQT2) in our two LQTS families, together with the finding that the P448R appears to be a common, ethnic-specific polymorphism, mutational analysis was extended to the other LQTS-causing genes resulting in the identification of distinct HERG missense mutations in each of these two families. Heterologous expression of P448R-KVLQT1 yielded normal, wild-type (WT) currents. In contrast, the two unique HERG mutations resulted in dominant-negative suppression of the WT HERG channel. Our study has profound implications for those engaged in genetic research. Importantly, one child of the original proband was initially diagnosed with LQT1 based upon the presence of P448R-KVLQT1 and was treated with beta-blockers. However, he did not possess the subsequently determined LQT2-causing mutation. On the other hand, his untreated P448R-negative brother harbored the true, disease-causing HERG mutation. These findings underscore the importance of distinguishing channel polymorphisms from mutations pathogenic for LQTS and emphasize the importance of using appropriate ethnically matched controls in the genotypic analysis of LQTS.

A negatively charged residue in the outer mouth of rat sodium channel determines the gating kinetics of the channel.

Previous studies using combined techniques of site-directed mutagenesis and electrophysiology of voltage-gated Na(+) channels have demonstrated that there are significant overlaps in the regions that are important for the two fundamental properties of the channels, namely gating and permeation. We have previously shown that a pore-lining residue, W402 in S5-S6 region (P loop) in domain I of the micro1 skeletal muscle Na(+) channel, was important in the gating of the channel. Here, we determined the role of an adjacent pore-lining negatively charged residue (E403) in channel gating. Charge neutralization or substitution with positively charged side chain at this position resulted in a marked delay in the rate of recovery from slow inactivation. Indeed, the fast inactivation process appeared intact. Restoration of the negatively charged side chain with a sulfhydryl modifier, MTS-ethylsulfonate, resulted in a reactivation profile from a slow-inactivated state, which was indistinguishable from that of the wild-type channels. We propose an additional functional role for the negatively charged residue. Assuming no major changes in the pore structure induced by the mutations, the negatively charged residue E403 may work in concert with other pore regions during recovery from slow inactivation of the channel. Our data represent the first report indicating the role of negative charge in the slow inactivation of the voltage-gated Na(+) channel.