New mode of Dunaliella biotechnology: two-phase growth for β-carotene production

Attempts to adapt the laboratory experience of bi-phasic growth and carotenogenesis in Dunaliella to large-scale conditions were highly successful. Algae were initially cultivated in stage one for optimizing biomass production of cells containing a low β-carotene to chlorophyll ratio. The culture was then transferred to stage two, diluted to about one third and induced for carotenogenesis. The bi-phase cultivation increased β- carotene productivity to 450 mg m^-2 d^-1 in stage one and to 300 mg m^-2 d^-1 in stage two, compared to the relatively low productivity of below 200 mg β- carotene m^-2 d^-1, in the conventional one phase type of cultivation. The results indicate that a selected algal product can be promoted specifically by growth manipulation of the alga and its environment.     

Interaction of the pBR 322-coded RTEM beta-lactamase with substrates. Evidence for specific conformational transitions.

The rate of inactivation of RTEM-1 beta-lactamase by Pronase is accelerated by class A ('resistant') penicillins. Other substrates (class S penicillin and cephalosporins) protect against the inactivation. Cefoxitin, a semi-synthetic cephamycin, induces a more extensive, hysteretic response. In its presence the enzyme is inactivated by trypsin as well as by Pronase.     

The miR-30 MicroRNA Family Targets smoothened to Regulate Hedgehog Signalling in Zebrafish Early Muscle Development

The importance of microRNAs in development is now widely accepted. However, identifying the specific targets of individual microRNAs and understanding their biological significance remains a major challenge. We have used the zebrafish model system to evaluate the expression and function of microRNAs potentially involved in muscle development and study their interaction with predicted target genes. We altered expression of the miR-30 microRNA family and generated phenotypes that mimicked misregulation of the Hedgehog pathway. Inhibition of the miR-30 family increases activity of the pathway, resulting in elevated ptc1 expression and increased numbers of superficial slow-muscle fibres. We show that the transmembrane receptor smoothened is a target of this microRNA family. Our results indicate that fine coordination of smoothened activity by the miR-30 family allows the correct specification and differentiation of distinct muscle cell types during zebrafish embryonic development.     

GPAT2, a mitochondrial outer membrane protein,in piRNA biogenesis in germline stem cells

piRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.     

Hsp90 restrains ErbB-2/HER2 signalling by limiting heterodimer formation

ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes.     

Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway

Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP-GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia.