Homologues of oxysterol-binding proteins affect Cdc42p- and Rho1p-mediated cell polarization in Saccharomyces cerevisiae

Polarized cell growth requires the establishment of an axis of growth along which secretion can be targeted to a specific site on the cell cortex. How polarity establishment and secretion are choreographed is not fully understood, though Rho GTPase- and Rab GTPase-mediated signaling is required. Superimposed on this regulation are the functions of specific lipids and their cognate binding proteins. In a screen for Saccharomyces cerevisiae genes that interact with Rho family CDC42 to promote polarity establishment, we identified KES1/OSH4, which encodes a homologue of mammalian oxysterol-binding protein (OSBP). Other yeast OSH genes (OSBP homologues) had comparable genetic interactions with CDC42, implicating OSH genes in the regulation of CDC42-dependent polarity establishment. We found that the OSH gene family (OSH1-OSH7) promotes cell polarization by maintaining the proper localization of septins, the Rho GTPases Cdc42p and Rho1p, and the Rab GTPase Sec4p. Disruption of all OSH gene function caused specific defects in polarized exocytosis, indicating that the Osh proteins are collectively required for a secretory pathway implicated in the maintenance of polarized growth.     

Deletion of OSH3 gene confers resistance against ISP-1 in Saccharomyces cerevisiae

Sphingolipids have been reported to regulate the growth and death of mammalian and yeast cells, but their precise mechanisms are unknown. In this paper, it was shown that the deletion of the oxysterol binding protein homologue 3 (OSH3) gene confers hyper resistance against ISP-1, an inhibitor of sphingolipid biosynthesis, in the yeast Saccharomyces cerevisiae. Furthermore, the overexpression of the ROK1 gene, which directly binds to Osh3p, conferred resistance against ISP-1, and the deletion of the KEM1 gene, which regulates microtubule functions, exhibited ISP-1 hypersensitivity. And yet, an ISP-1 treatment caused an abnormal mitotic spindle formation, and the ISP-1-induced cell cycle arrest was rescued by the deletion of the OSH3 gene. Taken together, it is suggested that the expression levels of the OSH3 gene influence the ISP-1 sensitivity of S. cerevisiae, and the sphingolipids are necessary for normal mitotic spindle formation in which the Osh3p may play a pivotal role.     

Isolation and characterization of a rice homebox gene, OSH15

In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem.     

Assessment of personal protective equipment use and occupational exposures in small industries in Jeddah: Health implications for workers

Small-scale industries account for a large proportion of jobs and play a vital role in most countries’ economic growth and prosperity. Due to the very low use of personal protective equipment (PPEs), employees are exposed to numerous physical, chemical, and accidental hazards in small-scale industries. PPEs are very effective in minimizing occupational injuries, accidents, and other hazards which otherwise result in substantial manpower and financial losses. The study objective was to assess the availability and use of PPEs as well as self-reported occupational exposures among workers in surveyed small industries in Jeddah. The study involved 102 workers from 28 small-scale industries (vehicle repair, welding, and paint). A survey was conducted to gather data of socio-demographic characteristics, self-reported occupational exposures, and frequency of PPEs used by workers. The occupational exposures (never exposed, sometimes exposed and always exposed) were reported in percentages including; noise exposure (19.6, 73.5 and 6.9%); dust/smoke exposure (9.8, 69.6 and 20.6%); vapors/fumes exposure (11.8, 60.8 and 27.5%); and direct sunlight (43.1, 56.9 and 0%), respectively. The reported use of different PPEs in descending order was; knee joints mats (50%), welding shields (50%), safety glasses (33.3%), gloves (27.5%), face masks (26.5%), safety shoes (10.8%) and earplugs/ muffs (8.8%). On the basis of this study findings, hand hygiene and general OSH awareness like interventions can be developed which will help in minimizing workplace exposures among small-scale industry workers.     

Two-hybrid cloning and characterization of OSH3, a yeast oxysterol-binding protein homolog

We identify Osh3p, one of seven yeast oxysterol-binding protein (OSBP) homologs, by its protein-protein interactions with a DEAD-box RNA helicase, Rok1p. The ROK1 gene was initially identified by its ability on a high-copy number plasmid to suppress the nuclear fusion defect caused by the kem1 null mutation. Our results show that OSH3 also affects nuclear fusion in a kem1-specific manner; the nuclear fusion defect of kem1 was intensified by the multicopy expression of OSH3. The Osh3p synthesis was highly induced by alpha-mating pheromone. We also found that OSH3 overexpression promoted filamentation growth of the Sigma1278b wild-type strain and suppressed the filamentation growth defect of the ste12 mutation. These results lead us to a new understanding of cellular functions of the yeast OSBPs.     

KNOX homeobox genes are sufficient in maintaining cultured cells in an undifferentiated state in rice

We produced transgenic rice calli, which constitutively express each of four KNOX family class 1 homeobox genes of rice, OSH1, OSH16, OSH15, and OSH71, and found that constitutive and ectopic expression of such genes inhibits normal regeneration from transformed calli, which showed continuous growth around their shoot-regenerating stages. Transgenic calli transferred onto regeneration medium began to display green spots, a sign of regeneration, but most of the transformants continued to propagate green spots at given stages. In the normal shoot-regeneration process of calli, expression of endogenous OSH1 was restricted in presumptive shoot-regenerating regions of calli and not observed in other areas. This restricted expression pattern should be required for further differentiation of the regenerating shoots. Thus our present results support the proposed function that KNOX family class 1 homeobox genes play a role in the formation and maintenance of the undetermined meristematic state of cells.     

The sterol-binding protein Kes1/Osh4p is a regulator of polarized exocytosis

Oxysterol-binding protein (OSBP)-related protein Kes1/ Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4(Y97F) is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein.