Cloning of an ovule specific promoter from Arabidopsis thaliana and expression of beta-glucuronidase

Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.     

Nimodipine is more effective than nifedipine in attenuating morphine tolerance on chronic co-administration in the rat tail-flick test

Opioids, when co-administered with L-type calcium channel blockers (L-CCBs) show morphine like higher antinociceptive effect. This antinociceptive effect has been further investigated using a different experimental paradigm. The effect of two different L-CCBs (nifedipine and nimodipine) on morphine-induced antinociception was studied by the tail-flick test (40 min after morphine administration) in adult Wistar rats. A fixed-dose of nimodipine or nifedipine (2 mg/kg, once daily) was combined with a fixed dose of morphine (10 mg/kg, twice daily) for 10 days. Co-administration of L-CCBs significantly increased the antinociceptive effect of morphine, even 12 hr after administration. Also, nimodipine was more effective than nifedipine. Nimodipine was further studied using a higher and escalating doses of morphine (20-30 mg/kg twice daily for 14 days). Nimodipine increased the antinociceptive effect of morphine in the latter part of the study (days nine to fourteen) though significant difference was observed on 11th evening and 12th morning. No obvious adverse effects were observed in the present study. The results show for the first time that nimodipine is more effective than nifedipine and that these L-CCBs continue to be effective, even 12 hr after administration in the tail-flick test.     

Stabilization of the pertussis toxin secretion apparatus by the C terminus of PtlD

Pertussis toxin (PT) is secreted from Bordetella pertussis by a type IV secretion system, known as the Ptl transporter, that comprises nine different proteins, PtlA to PtlI. In this study, we found that PtlD is required for the stability of three Ptl proteins, PtlE, PtlF, and PtlH. A region limited to the C-terminal 72 amino acids of PtlD (amino acids 392 to 463) was sufficient for maintaining the stability of PtlE, PtlF, and PtlH, although this region was not sufficient to support secretion of the toxin. Further analysis demonstrated that a stretch of 10 amino acids at the C-terminal end of PtlD (amino acids 425 to 434) contributes to transporter stability.     

Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial complex II in the post-ischemic myocardium

Increased O(2)* and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart ( Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282, 32640-32654 ). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide ((77)AAFGLSEAGFNTACVTK(93)) of the 70-kDa protein as a chimeric epitope incorporating a "promiscuous" T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.     

Adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality

Sepsis is a multifactorial, and often fatal, disorder typically characterized by widespread inflammation and immune activation with resultant endothelial activation. In the present study, we postulated that the adipokine adiponectin serves as a critical modulator of survival and endothelial activation in sepsis. To this aim, we evaluated both loss-of-function (adiponectin gene-deficient mice) and subsequent gain-of-function (recombinant adiponectin reconstitution) strategies in two well-established inflammatory models, cecal ligation perforation (CLP) and thioglyocollate-induced peritonitis. Adipoq(-/-) mice, subjected to CLP, exhibited a profound ( approximately 8-fold) reduction in survival compared with their wild-type Adipoq(+/+) littermates after 48 h. Furthermore, compared with wild-type controls, thioglycollate challenge resulted in a markedly greater influx of peritoneal neutrophils in Adipoq(-/-) mice accompanied by an excess production of key chemoattractant cytokines (IL-12p70, TNFalpha, MCP-1, and IL-6) and upregulation of aortic endothelial adhesion molecule VCAM-1 and ICAM-1 expressions. Importantly, all of these effects were blunted by recombinant total adiponectin administration given 3 days prior to thioglycollate challenge. The protective effects of adiponectin were ascribed largely to higher-order adiponectin oligomers, since administration of recombinant C39A trimeric adiponectin did not attenuate endothelial adhesion molecule expression in thioglycollate-challenged Adipoq(-/-) mice. These data suggest a critical role of adiponectin as a modulator of survival and endothelial inflammation in experimental sepsis and a potential mechanistic link between adiposity and increased sepsis.     

Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus interacts with origin recognition complexes at the LANA binding sequence within the terminal repeats

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the cis-acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G(1)/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.     

Mapping QTLs for popping ability in a popcorn × flint corn cross

Popping expansion volume (PEV) in popcorn (Zea mays L.) is a distinct heritable character and defined as the ratio of the volume after popping to the volume before popping. PEV is quantitatively inherited and 3–4 genes/quantitative trait loci (QTLs) have been implicated. In the present study, we have dissected the quantitative PEV into two component traits, viz., flake volume (FV) and percent unpopped kernels (UPK), and mapped QTLs using SSR markers for all three traits with 194 F₃ families derived from a popcorn (A-1-6) × flint corn (V273) cross. Heritability (broad sense) estimates for PEV, FV and UPK based on F₃ mean bases were 0.72, 0.54 and 0.68, respectively. The QTL analyses for the three traits based on combined environment data were performed by composite interval mapping using QTL cartographer. Four QTLs were identified for PEV on chromosomes 1, 3, 8 and 10, which together explained 62% of the phenotypic variance (σ²p). Four QTLs were found on chromosomes 1, 5, 9 and 10 for FV (explaining 44% of σ²p) and five QTLs for UPK on chromosomes 1, 3, 4, 5 and 9 (explaining 57% of σ²p). The relative efficiency estimates of marker-based selection in comparison to phenotypic selection for PEV (1.10), FV (1.22) and UPK (1.11) indicated that marker-based selection could be relatively more efficient. The QTL on chromosome 1S for PEV was found to be most significant, where QTLs for hard endosperm starch concentration had been detected earlier. A. Kumar and H.S. Rao contributed equally to this research work.     

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated ‘126-1’, was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F₁s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for ‘126-1’ cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of ‘126-1’ cytoplasm for hybrid seed production. The uniqueness of ‘126-1’ has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.     

Effect of tree leaf as supplementation on nutrient digestion and rumen fermentation pattern in sheep grazing semi-arid range of India – II

A study was conducted to evaluate the effects of supplementing with different tree leaves on nutrient digestion, rumen fermentation and blood parameters of sheep. Thirty adult Malpura rams (39.0 ± 0.56 kg) were divided into five groups of six each. They were grazed as a single flock on a semi-arid rangeland and after the end of routine grazing period (08:00–17:00 h), first group (G1), which was not provided with any supplementation, served as control group. Second group (G2) was supplemented with 200 g of a conventional concentrate mixture per head per day, whereas third, fourth and fifth groups (G3, G4 and G5) were supplemented with approximately 200 g dry matter (DM) per day freshly cut foliage from Ailanthes excelsa, Azardirachta indica and Bauhinia racemosa, respectively. Protein content (g kg⁻¹ DM) in A. excelsa, A. indica and B. racemosa foliage was 197, 128 and 132, respectively. A. indica and B. racemosa foliages also contained 123.2 and 211.2 g kg⁻¹ DM condensed tannin (CT) with protein precipitating capacity (PPC) of 16.5 and 46.5 g kg⁻¹ DM. None of the tree leaves contained hydrolysable tannin (HT). Dry matter intake (DMI, g day⁻¹) was 591, 766, 865, 974 and 939 in G1, G2, G3, G4 and G5, respectively. Digestible crude protein (DCP) and metabolisable energy (ME) intakes in supplemented groups G2–G5 were higher (P < 0.05) compared to control (G1). Supplementation improved digestibility of all nutrients in all groups. Rumen fermentation study indicated lower (P < 0.05) ammonia and total N in the rumen liquor collected from G5 sheep compared to the other supplemented groups. Although haemoglobin (Hb, g dl⁻¹) levels showed small changes among groups, blood urea nitrogen (BUN, mg dl⁻¹) was lowest in G5 compared to the other groups. Initial BW were similar among the groups. After 60 days of experimental feeding, all animals maintained their BW, except sheep in the control group (G1), which lost BW. Results indicate that for adult sheep grazing on a semi-arid range, supplementation with a concentrate mixture could be replaced by tree leaves like A. excelsa, A. indica and B. racemosa, during the lean season to maintain their BW. In addition, supplementing with tree leaves containing condensed tannin has advantages in terms of N utilization.