Abscisic acid catabolism enhances dormancy release of grapevine buds

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)‐mediated repression of bud–meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up‐regulation of VvA8H‐CYP707A4, which encodes ABA 8′‐hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H‐CYP707A4 within grape buds, (b) the VvA8H‐CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H‐CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H‐CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H‐CYP707A4 level in the bud is up‐regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA‐mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.     

Rhythms in acoustical communication by the oriental hornet, vespa orientalis

By analysis of the acoustical spectrum within the nest of the oriental hornet, three distinct and characteristic categories of sounds have been recognized: (a) Hunger signals produced by the hungry larvae through the scraping of their mandibles against the comb cell walls; (b) sounds which the workers, arranged in a ‘resting circle’ around the queen, produce by tapping their abdomens on the surfaces of the cells. These sounds are temporarily designated as ‘the taps of workers facing the queen’; and (c) additional worker sounds, also produced as in (b) but of a different rhythm, which have been designated in earlier publications as the ‘awakening taps of workers’ (Ishay & Schwartz 1965; Schaudeinischley & Ishay 1968; Ishay & Landau 1972; Ishay & Schwartz 1973). The particular ‘beat’ (spcificity) of each of the three sound groups has been investigated in an attempt to understand the significance of rhythms as a means of communication among hornets.     

Isolation and characterization of the ccmM gene required by the cyanobacterium Synechocystis PCC6803 for inorganic carbon utilization

A high CO₂-requiring mutant of Synechocystis PCC6803 (G3) capable of C_i transport but unable to utilize the intracellular C_i pool for photosynthesis was constructed. A DNA clone of 6.1 kbp that transforms the G3 mutant to the wild-type phenotype was isolated from a Synechocystis PCC6803 genomic library. Complementation test with subclones allocated the mutation site within a DNA fragment of 674 bp nucleotides. Sequencing analysis of the mutation region elucidated an open reading frame encoding a 534 amino-acid protein with a significant sequence homology to the protein coded by the ccmN gene of Synechococcus PCC7942. The ccmM-like gene product of Synechocystis PCC6803 contains four internal repeats with a week similarity to the rbcS gene product. An open reading frame homologous to the ccmN gene of Synechococcus PCC7942 was found downstream to the ccmM-like gene. As opposed to the Synechococcus PCC7942 ccmM and ccmN genes located 2 kbp upstream to, and oriented in the same direction as, the rbc operon, the ccm-like genes in Synechocystis PCC6803 are not located within 22 kbp upstream to the rbcL gene of the Rubisco operon. Thus, despite the resemblance in clustering of the ccmM and ccmN genes in both cyanobacterial species, the difference in their genomic location relative to the rbc genes demonstrates variability in structural organization of the genes involved in inorganic carbon acquisition.Abbreviations CCM CO₂-concentrating mechanism - C_i inorganic carbon - HCR high CO₂-requiring - kbp kilobase pair - ORF open reading frame - Rubisco ribulose 1,5-bisphosphate carboxylase-oxygenase gene - SSC sodium chloride and sodium citrate - WT wild-type     

Radiotherapy combined with gemcitabine and oxaliplatin in pancreatic cancer cells

Clinical evidence suggests that gemcitabine (Gem) plus oxaliplatin (Ox) is superior to gemcitabine alone in advanced pancreatic carcinoma. The addition of radiation to gemcitabine improves response and is a standard treatment for locally advanced disease. We investigated the effect of oxaliplatin on gemcitabine-based chemoradiation by determining whether gemcitabine and oxaliplatin produced synergistic cytotoxicity using median effect analysis and radiosensitization using clonogenic survival assays. We analyzed the effects of gemcitabine and oxaliplatin on cell cycle distribution by DNA content and on radiation-induced DNA damage repair by phosphorylated H2AX (gamma-H2AX). Gemcitabine and oxaliplatin produced schedule-dependent synergistic cytotoxicity in BxPC-3 and Panc-1 cells (combination indices: 0.76 +/- 0.05, 0.61 +/- 0.11). In BxPC-3 cells, oxaliplatin did not affect gemcitabine-mediated radiosensitization (Gem 1.99 +/- 0.27; Gem + Ox 2.38 +/- 0.30). In Panc-1 cells, oxaliplatin significantly enhanced gemcitabine-mediated radiosensitization (Gem 1.31 +/- 0.05; Gem + Ox 2.90 +/- 0.31). Radiosensitization by gemcitabine was accompanied by early S-phase arrest and induction/persistence of gamma-H2AX protein, which were unaltered by oxaliplatin. Addition of oxaliplatin to gemcitabine produces radiosensitization equal to or greater than gemcitabine alone, supporting our clinical investigation of oxaliplatin with gemcitabine-radiation in pancreatic cancer aimed at improving systemic disease control while maintaining local tumor radiosensitization.     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type     

Versatile role of heparanase in inflammation

Heparanase is the only known mammalian endoglycosidase capable of degrading heparan sulfate glycosaminoglycan, both in extracellular space and within the cells. It is tightly implicated in cancer progression and over the past few decades significant progress has been made in elucidating the multiple functions of heparanase in malignant tumor development, neovascularization and aggressive behavior. Notably, current data show that in addition to its well characterized role in cancer, heparanase activity may represent an important determinant in the pathogenesis of several inflammatory disorders, such as inflammatory lung injury, rheumatoid arthritis and chronic colitis. Nevertheless, the precise mode of heparanase action in inflammatory reactions remains largely unclear and recent observations suggest that heparanase can either facilitate or limit inflammatory responses, when tissue/cell-specific contextual cues may dictate an outcome of heparanase action in inflammation. In this review the involvement of heparanase in modulation of inflammatory reactions is discussed through a few illustrative examples, including neuroinflammation, sepsis-associated lung injury and inflammatory bowel disease. We also discuss possible action of the enzyme in coupling inflammation and tumorigenesis in the setting of inflammation-triggered cancer.     

The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs,targets lymphocytes including activated B cells,and supports antibody responses to a bacterial pathogen in vivo

Monokine induced by IFN-gamma (Mig; CXC chemokine ligand 9) is an IFN-gamma-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4(+) and CD8(+), and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig(-/-) mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig(-/-) mice showed reductions of 50-75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-gamma induced the expression of mumig in APCs, including CD8 alpha(+) and CD8 alpha(-) dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.     

Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synechocystis PCC 6803 via targeted gene recombination.

Summary The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3-truncated gene copy into the psbA-I locus. Both rbcL genes, in the psbA-I region and at the rbc locus, were non-functional; The former due to the 3 truncation, and the latter due to a deletion in the 5-region (creating a 5 truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO₂-requiring mutant (cyanorubrum). The 3 and the 5 truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L₈S₈) and R. rubrum L₂) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.