Calcium dependence of proteinase-activated receptor 2 and cholecystokinin-mediated amylase secretion from pancreatic acini

Pancreatic acini secrete digestive enzymes in response to a variety of secretagogues including CCK and agonists acting via proteinase-activated receptor-2 (PAR2). We employed the CCK analog caerulein and the PAR2-activating peptide SLIGRL-NH(2) to compare and contrast Ca(2+) changes and amylase secretion triggered by CCK receptor and PAR2 stimulation. We found that secretion stimulated by both agonists is dependent on a rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and that this rise in [Ca(2+)](i) reflects both the release of Ca(2+) from intracellular stores and accelerated Ca(2+) influx. Both agonists, at low concentrations, elicit oscillatory [Ca(2+)](i) changes, and both trigger a peak plateau [Ca(2+)](i) change at high concentrations. Although the two agonists elicit similar rates of amylase secretion, the rise in [Ca(2+)](i) elicited by caerulein is greater than that elicited by SLIGRL-NH(2). In Ca(2+)-free medium, the rise in [Ca(2+)](i) elicited by SLIGRL-NH(2) is prevented by the prior addition of a supramaximally stimulating concentration of caerulein, but the reverse is not true; the rise elicited by caerulein is neither prevented nor reduced by prior addition of SLIGRL-NH(2). Both the oscillatory and the peak plateau [Ca(2+)](i) changes that follow PAR2 stimulation are prevented by the phospholipase C (PLC) inhibitor U73122, but U73122 prevents only the oscillatory [Ca(2+)](i) changes triggered by caerulein. We conclude that 1) both PAR2 and CCK stimulation trigger amylase secretion that is dependent on a rise in [Ca(2+)](i) and that [Ca(2+)](i) rise reflects release of calcium from intracellular stores as well as accelerated influx of extracellular calcium; 2) PLC mediates both the oscillatory and the peak plateau rise in [Ca(2+)](i) elicited by PAR2 but only the oscillatory rise in [Ca(2+)](i) elicited by CCK stimulation; and 3) the rate of amylase secretion elicited by agonists acting via different types of receptors may not correlate with the magnitude of the [Ca(2+)](i) rise triggered by those different types of secretagogue.     

Purification and characterization of a highly active chromate reductase from endophytic Bacillus sp. DGV19 of Albizzia lebbeck (L.) Benth. actively involved in phytoremediation of tannery effluent-contaminated sites

Phytoremediation using timber-yielding tree species is considered to be the most efficient method for chromium/tannery effluent-contaminated sites. In this study, we have chosen Albizzia lebbeck, a chromium hyperaccumulator plant, and studied one of its chromium detoxification processes operated by its endophytic bacterial assemblage. Out of the four different groups of endophytic bacteria comprising Pseudomonas, Rhizobium, Bacillus, and Salinicoccus identified from A. lebbeck employed in phytoremediation of tannery effluent-contaminated soil, Bacillus predominated with three species, which exhibited not only remarkable chromium accumulation ability but also high chromium reductase activity. A chromate reductase was purified to homogeneity from the most efficient chromium accumulator, Bacillus sp. DGV 019, and the purified 34.2-kD enzyme was observed to be stable at temperatures from 20°C to 60°C. The enzyme was active over a wide range of pH values (4.0–9.0). Furthermore, the enzyme activity was enhanced with the electron donors NADH, followed by NADPH, not affected by glutathione and ascorbic acid. Cu²⁺ enhanced the activity of the purified enzyme but was inhibited by Zn²⁺ and etheylenediamine tetraacetic acid (EDTA). In conclusion, due to its versatile adaptability the chromate reductase can be used for chromium remediation.     

Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage

Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn²⁺ finger motifs in the CTD. The Zn²⁺ finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn²⁺ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn²⁺ fingers from the mycobacterial topoI could be associated with Zn²⁺ export and homeostasis.     

Analysis of heterosis and recombination loss for fitness and productivity traits in different hybrids of mulberry silk moth Bombyx mori

Three different races of lepidopteron silk moth Bombyx mori were used in reciprocal and inter se crosses to determine heterosis effects at F₁ and recombination loss at the F₂ generation for three fitness traits (fecundity, larval duration, survival rate) and four productivity traits (larval weight, cocoon weight, shell weight, filament length). Eleven mating types were represented in the present study, including three pure breeds and a variety of F₁ and F₂ populations arising from regular and reciprocal crosses, respectively. Equations were derived to evaluate heterosis, maternal and overdominance effects for the above traits. Estimates of heterosis and overdominance effects revealed significant heterosis effects for all the traits, but overdominance was only seen for larval duration (favorable effect) and survival rate (unfavorable effect). Maternal effects were significant for the majority of the traits under study. The results revealed significant reduction for all the quantitative traits from F₁ to F₂, except for larval duration. The most obvious explanation for the reduction of fitness parameters and productive traits is the reduction in heterozygosity from F₁ to F₂ (it is expected that one half of the heterozygosity of F₁ is lost in F₂). For larval duration this explanation seems insufficient and breakdown of epistatic gene effects (i.e. recombination loss) has been suggested.     

Age dependent alterations in phospholipid composition of a saprophytic and a pathogenic strain of Nocardia

Nocardia polychromogenes (saprophytic) and Nocardia asteroides (pathogenic) showed characteristic patterns in changes of cellular lipids during growth. Total lipids and total phospholipids decreased with the age of the culture in the saprophytic strain, whereas in the pathogenic strain total lipids increased throughout the culture period and the total phospholipids decreased in the late stationary phase. The decrease in total phospholipids in saprophytic strain was reflected in the individual phosphatides. In the pathogenic strain, the phosphatidylinositomannoside content doubled in early stationary phase. Differences were observed in fatty acid composition of phosphatides at various stages of growth, but the ratio of saturated to unsaturated fatty acids remained unaltered.     

Elevated CO2 increases water use efficiency by sustaining photosynthesis of water-limited maize and sorghum

Maize and grain sorghum seeds were sown in pots and grown for 39 days in sunlit controlled-environment chambers at 360 (ambient) and 720 (double-ambient, elevated) μmol mol⁻¹ carbon dioxide concentrations [CO₂]. Canopy net photosynthesis (PS) and evapotranspiration (TR) was measured throughout and summarized daily from 08:00 to 17:00 h Eastern Standard Time. Irrigation was withheld from matched pairs of treatments starting on 26 days after sowing (DAS). By 35 DAS, cumulative PS of drought-stress maize, compared to well-watered plants, was 41% lower under ambient [CO₂] but only 13% lower under elevated [CO₂]. In contrast, by 35 DAS, cumulative PS of drought-stress grain sorghum, compared to well-watered plants, was only 9% lower under ambient [CO₂] and 7% lower under elevated [CO₂]. During the 27-35 DAS drought period, water use efficiency (WUE, mol CO₂ Kmol⁻¹ H₂O), was 3.99, 3.88, 5.50, and 8.65 for maize and 3.75, 4.43, 5.26, and 9.94 for grain sorghum, for ambient-[CO₂] well-watered, ambient-[CO₂] stressed, elevated-[CO₂] well-watered and elevated-[CO₂] stressed plants, respectively. Young plants of maize and sorghum used water more efficiently at elevated [CO₂] than at ambient [CO₂], especially under drought. Reductions in biomass by drought for young maize and grain sorghum plants were 42 and 36% at ambient [CO₂], compared to 18 and 14% at elevated [CO₂], respectively. Results of our water stress experiment demonstrated that maintenance of relatively high canopy photosynthetic rates in the face of decreased transpiration rates enhanced WUE in plants grown at elevated [CO₂]. This confirms experimental evidence and conceptual models that suggest that an increase of intercellular [CO₂] (or a sustained intercellular [CO₂]) in the face of decreased stomatal conductance results in relative increases of growth of C₄ plants. In short, drought stress in C₄ crop plants can be ameliorated at elevated [CO₂] as a result of lower stomatal conductance and sustaining intercellular [CO₂]. Furthermore, less water might be required for C₄ crops in future higher CO₂ atmospheres, assuming weather and climate similar to present conditions.     

Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery

BACKGROUND: The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety. METHODS: Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently. RESULTS: Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone. CONCLUSIONS: Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes, generated in this study have considerable potential to facilitate DNA delivery and enhance transfection. The domains in these fusion proteins would be promising in the development of non-viral gene delivery vectors particularly in cells that do not divide.     

Changes in cholesterol levels in the plasma membrane modulate cell signaling and regulate cell adhesion and migration on fibronectin

The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton.     

Fibronectin and laminin enhance engraftibility of cultured hematopoietic stem cells

To test the hypothesis that extracellular matrix (ECM) components maintain stem cell property, murine bone marrow (BM) cells were expanded in fibronectin and laminin coated plate in the presence of cytokines. We observed significant phenotypic and functional improvement of expanded cells. In 10 days, 800-fold expansion of colony-forming unit-granulocyte erythrocyte monocyte megakaryocyte (CFU-GEMM) was observed in the cultured cells. No apparent activation of cell cycle was observed, but CD29 and very late antigen-4 (VLA-4) expression was increased, as compared to the normal BM cells. A fraction of the expanded cells became verapamil sensitive, suggesting upregulation of multi-drug resistant gene(s), as found in the primitive hematopoietic stem cells (HSCs). Competitive repopulation assay confirmed that HSCs compartment was amplified during culture. Overall, our study clearly demonstrated that ex vivo culture of murine HSCs in the presence of fibronectin and laminin resulted in expansion of primitive stem cells and improvement in the marrow engraftibility.     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.