Enhancing effects of 24-epibrassinolide and Putrescine on the antioxidant capacity and free radical scavenging activity of <Emphasis Type="Italic">Raphanus sativus</Emphasis> seedlings under Cu ion stress

The present investigation recorded significant restoration of seedling growth (root length, shoot length and fresh weight) upon application of 24-epibrassinolide (EBL) and putrescine (Put) to 7-day-old seedlings of Raphanus sativus L. cv. Pusa chetki grown under copper (Cu) ion stress. EBL and Put with/or without Cu ion treated seedlings showed increased titers of ascorbic acid, total phenols and proline when compared with Cu-stressed seedlings. Differential responses in the activities of guaiacol peroxidase (GPOX) and catalase (CAT) were noted for EBL and Put alone or with/or without Cu ion treatment. Decreased activities of glutathione reductase (GR) and superoxide dismutase (SOD) noted for EBL and Put alone were observed to enhance significantly when applied in combination with Cu ion solution. A remarkable decrease in malondialdehyde contents was observed in seedlings treated with EBL and Put alone and with/or without Cu ion stress. Enhanced free radical scavenging activities were also recorded for seedlings given EBL and Put alone or in combination over Cu ion stressed seedlings. Maximum DPPH activity was observed in seedlings treated with Put and EBL 10⁻⁹ M + Put. Significant enhancements in deoxyribose and reducing power activities were too recorded for Put, EBL and Put + 10⁻⁹ M EBL treatments. Improved seedling growth, antioxidant levels (ascorbic acid, total phenols and proline) and enzymic (GPOX, CAT, SOD and GR) activities and free radical scavenging capacities along with reduced membrane damage in seedlings given EBL and Put with/or without Cu stress suggests significant and positive interactions of EBL and Put in alleviating the Cu ion induced oxidative damage in radish seedlings.     

S1P lyase: a novel therapeutic target for ischemia-reperfusion injury of the heart

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic preconditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible S1P catabolism. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, we evaluated SPL inhibition as a strategy for reducing cardiac ischemia-reperfusion (I/R) injury. We measured SPL expression and enzyme activity in murine hearts. Basal SPL activity was low in wild-type cardiac tissue but was activated in response to 50 min of ischemia (n = 5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size, and increased functional recovery after I/R compared with littermate controls (n = 5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is a Federal Drug Administration-approved food additive that inhibits SPL. When given overnight at 25 mg/l in drinking water, THI raised S1P levels and reduced SPL activity (n = 5, P < 0.01). THI reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and to 40 min of reperfusion in ex vivo hearts (n = 7, P < .01). These data correlated with an increase in MAP kinase-interacting serine/threonine kinase 1, eukaryotic translation initiation factor 4E, and ribosomal protein S6 phosphorylation levels after I/R, suggesting that SPL inhibition enhances protein translation. Pretreatment with an S1P? and S1P? receptor antagonist partially reversed the effects of THI. These results reveal, for the first time, that SPL is an ischemia-induced enzyme that can be targeted as a novel strategy for preventing cardiac I/R injury.     

Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C₃₀ carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.     

Crossability of Brassica tournefortii and B. rapa, and morphology and cytology of their F1 hybrids

The crossability between Brassica tournefortii (TT, 2n = 20) and Brassica rapa (AA, 2n = 20) and the cytomorphology of their F₁ hybrids were studied. Hybrids between these two species were obtained only when B. tournefortii was involved as a female parent. The hybrid plants were intermediate for most of the morphological attributes and were found to be free from white rust under field conditions. The F₁ plants showed poor pollen fertility, although occasional seed set was achieved from open pollination. Self-pollination or backcrosses did not yield any seeds in these plants. The occurrence of chromosome association ranging from bivalents (0–7), trivalents (0–2) to a rare quadrivalent (0–1) in the dihaploid hybrids indicates pairing between the T and A genomes. The homoeologous pairing coupled with seed set in the F₁ plants offer an opportunity for interspecific gene transfers from B. tournefortii to B. rapa and vice-versa through interspecific hybridization. Received: 3 July 2000 / Accepted: 22 September 2000     

Antifungal and antibacterial activities of Taxus wallichiana Zucc

Current study was undertaken to evaluate the in vitro antifungal and antibacterial potential of methanol extract and subsequent fractions obtained after partitioning in organic solvents with variable polarity of the aerial parts of the tree Taxus wallichiana Zucc. Traditionally, this plant is often used in folk medicines in Pakistan for treating microbial infections. In order to rationalize the traditional use, methanol extracts of leaf, bark, and heartwood of Taxus wallichiana Zucc. were tested against six bacteria and six fungal strains using the Hole diffusion and macro-dilution methods. All extracts and fractions displayed significant antimicrobial effect. Only three fungal strains, Trichophyton longifusus, Microspoum canis, and Fusarium solani were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. In case of bacterial strains, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. Comparison results were carried out using imipinem, miconazole and amphotericin B as standard antibiotics.     

Elucidation of the anticancer potential and tubulin isotype-specific interactions of β-sitosterol

Beta-sitosterol (β-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the ‘SITO-site’) compared to the colchicine binding site at the interface of α- and β-tubulin. In this study, we investigated the anticancer efficacy of β-SITO against invasive breast carcinoma using MCF-7 cells. Since ‘isotypes’ of β-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of β-tubulin in αβ-tubulin dimer. β-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of β-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to β-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that β-SITO preferentially binds with specific β-tubulin isotypes (β_II and β_III) in the αβ-tubulin dimer. Both these β-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of β-SITO, and its potential clinical utility against β_II and β_III isotype-overexpressing neoplasms.     

Absence of TGFβ signaling in embryonic vascular smooth muscle leads to reduced lysyl oxidase expression,impaired elastogenesis,and aneurysm

To address the requirement for TGFβ signaling in the formation and maintenance of the vascular matrix, we employed lineage‐specific mutation of the type II TGFβ receptor gene (Tgfbr2) in vascular smooth muscle precursors in mice. In both neural crest‐ and mesoderm‐derived smooth muscle, absence of TGFβ receptor function resulted in a poorly organized vascular elastic matrix in late‐stage embryos which was prone to dilation and aneurysm. This defect represents a failure to initiate formation of the elastic matrix, rather than a failure to maintain a preexisting matrix. In mutant tissue, lysyl oxidase expression was substantially reduced, which may contribute to the observed pathology. genesis 47:115–121, 2009. © 2009 Wiley‐Liss, Inc.     

Multimodal approach to explore the pathogenicity of BARD1, ARG 658 CYS,and ILE 738 VAL mutants

BARD1–BRCA1 complex plays an important role in DNA damage repair, apoptosis, chromatin remodeling, and other important processes required for cell survival. BRCA1 and BARD1 heterodimer possess E3 ligase activity and is involved in genome maintenance, by functioning in surveillance for DNA damage, thereby regulating multiple pathways including tumor suppression. BRCT domains are evolutionary conserved domains present in different proteins such as BRCA1, BARD1, XRCC, and MDC1 regulating damage response and cell-cycle control through protein–protein interactions. Nonetheless, the role of BARD1BRCT in the recruitment of DNA repair mechanism and structural integrity with BRCA1 complex is still implicit. To explicate the role of BARD1BRCT in the DNA repair mechanism, in silico, in vitro, and biophysical approach were applied to characterize BARD1 BRCT wild-type and Arg658Cys and Ile738Val mutants. However, no drastic secondary and tertiary structural changes in the mutant proteins were observed. Thermal and chemical denaturation studies revealed that mutants Arg658Cys and Ile738Val have a decrease in T_m and ?G than the wild type. In silico studies of BARD1 BRCT (568-777) and mutant protein indicate loss in structural compactness on the Ile738Val mutant. Comparative studies of wild-type and mutants will thus be helpful in understanding the basic role of BARD1BRCT in DNA damage repair.