The identification and characterization of osmotolerant yeast isolates from chemical wastewater evaporation ponds.

Ramat Hovav is a major chemical industrial park manufacturing pharmaceuticals, pesticides, and various aliphatic and aromatic halogens. All wastewater streams are collected in large evaporation ponds. Salinity in the evaporation ponds fluctuates between 3% (w/v) and saturation and pH values range between 2.0 and 10.0. We looked for microorganisms surviving in these extreme environmental conditions and found that 2 yeast strains dominate this biotope. 18S rDNA sequence analysis identified the isolates as Pichia guilliermondii and Rhodotorula mucilaginosa. Both isolates grew in NaCl concentrations ranging up to 3.5 M and 2.5 M, respectively, and at a pH range of 2-10. There was a distinct difference between the Rhodotorula and Pichia strains and S. cerevisiae RS16 that served as a control strain with respect to accumulation of osmoregulators and internal ion concentrations when exposed to osmotic stress. The Pichia and Rhodotorula strains maintained high glycerol concentration also in media low in NaCl. Utilization of various carbon sources was examined. Using a tetrazolium-based assay we show that the Rhodotorula and Pichia strains are capable of utilizing a wide range of different carbon sources including anthracene, phenanthrene, and other cyclic aromatic hydrocarbons.

Desert dust suppressing precipitation: a possible desertification feedback loop.

The effect of desert dust on cloud properties and precipitation has so far been studied solely by using theoretical models, which predict that rainfall would be enhanced. Here we present observations showing the contrary; the effect of dust on cloud properties is to inhibit precipitation. Using satellite and aircraft observations we show that clouds forming within desert dust contain small droplets and produce little precipitation by drop coalescence. Measurement of the size distribution and the chemical analysis of individual Saharan dust particles collected in such a dust storm suggest a possible mechanism for the diminished rainfall. The detrimental impact of dust on rainfall is smaller than that caused by smoke from biomass burning or anthropogenic air pollution, but the large abundance of desert dust in the atmosphere renders it important. The reduction of precipitation from clouds affected by desert dust can cause drier soil, which in turn raises more dust, thus providing a possible feedback loop to further decrease precipitation. Furthermore, anthropogenic changes of land use exposing the topsoil can initiate such a desertification feedback process.

Leukaemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro.

The cytokine leukaemia inhibitory factor (LIF) is up-regulated in glial cells after injury to the peripheral and central nervous systems. In addition, LIF is required for the changes in neuropeptide expression that normally occur when the axons of sympathetic and sensory neurons are transected. We investigated whether LIF is also necessary for the initial inflammatory response that follows mechanical injury to the sciatic nerve and cerebral cortex of adult mice. We find that inflammatory cell infiltration into crushed sciatic nerve is significantly slower in LIF knock-out (KO) mice compared with wild-type (WT) mice. Similarly, the microglial and astroglial responses to surgical injury of the cortex are significantly slower in LIF KO mice compared with WT mice. Consistent with these in vivo results, LIF is chemotactic for peritoneal macrophages in a microchamber culture assay. Thus, LIF is a key regulator of neural injury in vivo, where it is produced by glia and can act directly on neurons, glia and inflammatory cells. We also find that the initial inflammatory response to cortical injury is diminished in interleukin (IL)-6 KO mice. Surprisingly, however, the inflammatory response in LIF-IL-6 double KO mice is very similar to that of the single KO mice, suggesting that these cytokines may act in series rather than in parallel in this response.

An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells in vitro and in vivo.

Activation of the endothelin receptor B (ETRB) in cultured melanocyte precursors promotes cell proliferation while inhibiting differentiation, two hallmarks of malignant transformation. We therefore tested whether ETRB has a similar role in malignant transformation of melanoma. When tested in culture, we find that the selective ETRB antagonist BQ788 can inhibit the growth of seven human melanoma cell lines, but not a human kidney cell line. This inhibition often is associated with increases in pigmentation and in the dendritic shape that is characteristic of mature melanocytes. In three cell lines we also observe a major increase in cell death. In contrast, the endothelin receptor A (ETRA) antagonist BQ123 does not have these effects, although all the cell lines express both ETRA and ETRB mRNA. Extending these studies in vivo, we find that administration of BQ788 significantly slows human melanoma tumor growth in nude mice, including a complete growth arrest in half of the mice treated systemically. Histological examination of tumor sections suggests that BQ788 also enhances melanoma cell death in vivo. Thus, ETRB inhibitors may be beneficial for the treatment of melanoma.

Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro.

Genetic data in the mouse have shown that endothelin 3 (ET3) and its receptor B (ETRB) are essential for the development of two neural crest (NC) derivatives, the melanocytes and the enteric nervous system. We report here the effects of ET3 in vitro on the differentiation of quail trunk NC cells (NCC) in mass and clonal cultures. Treatment with ET3 is highly mitogenic to the undifferentiated NCC population, which leads to expansion of the population of cells in the melanocytic, and to a lesser extent, the glial lineages. The effect of ET3 on these two NC derivatives was confirmed by the quantitative analysis of clones derived from individual NCC subjected to ET3: we found a large increase in the survival and proliferation of unipotent and bipotent precursors for glial cells and melanocytes, with no significant effect on multipotent cells generating neurons. ET3 first stimulates expression of both ETRB and ETRB2 by cultured NCC. Then, under prolonged exposure to ET3, ETRB expression decreases and switches toward an ETRB2-positive melanogenic cell population. We therefore propose that the present in vitro experiments (long-lasting exposure to a high concentration of ET3) mimic the environment encountered by NCC in vivo when they migrate to the skin under the ectoderm that expresses ET3.

Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture.

Mutations in the endothelin 3 (EDN3) gene severely affect the development of neural crest-derived melanocytes. In this paper, we report the action of EDN3 on neural crest cells in vitro. The presence of EDN3 leads to a large increase in the number of cells, the majority of which eventually differentiate into melanocytes that aggregate to form a reproducible pigmentation pattern. Quantitative analysis of the effect of different culture conditions revealed that EDN3 initially promotes neural crest cell proliferation. This phase of expansion, which can be prolonged for a few weeks if the cells are replaced regularly, is followed by both a decrease in cell proliferation and the onset of melanocytic differentiation. Therefore, EDN3 is a potent mitogen for early neural crest cell precursors that can give rise to melanocytes.

Steel and c-kit in the development of avian melanocytes: a study of normally pigmented birds and of the hyperpigmented mutant silky fowl.

We describe here the expression of c-kit and Steel (Sl) genes during the development of melanocytes in normally pigmented strains of chick and quail compared to unpigmented (White Leghorn) and hyperpigmented (Silky Fowl) strains of chickens. By using the quail/chick chimera system, we found that the neural crest cells, which migrate dorso-laterally in the subectodermal mesenchyme to give rise to the melanocytes, express c-kit as early as E4, that is about 2 days after they have left the neural primordium. The Sl gene is expressed from E4 onward in the epidermis but not at all in the dermis at any developmental stage. As feather buds develop, Sl mRNA becomes restricted to the apical region of the feather filaments. During formation of the barbs and barbules of the down feather, production of the Steel factor is restricted to the external epidermal cells of the barbules. The cell bodies of the c-kit-positive melanocytes are then located in the internal border of the epidermal ridges and extend their processes toward the source of the Steel factor. We propose that the spatial restriction of Sl gene activity at that stage accounts for the morphology of the melanocytes and their vectorial secretion of melanin to the external barbule cells. As a whole, these results show that during skin development c-kit positive cells are present in the Steel factor-producing areas at the time when melanoblasts proliferate and differentiate. Interestingly, in the mouse, previous studies showed that the Sl gene is activated in the dermis where melanoblasts undergo most of their expansion (Nishikawa et al. [1991] EMBO J. 10:2111-2118). In the unpigmented and hyperpigmented mutants that we studied, expression of the Sl message, as judged quantitatively in Northern blots (for the SF embryos) or spatially by in situ hybridization, is similar to that observed in normal birds. In SF embryos the c-kit expressing melanoblasts migrate initially in the dorso-lateral migration pathway as in normal birds. However their number increases considerably in the dermis from E5 onward. From E7, they invade mesodermally derived organs that do not express the Sl gene. This suggests that another, still unknown, factor(s) is responsible for the survival, the proliferation, and the extensive spreading of melanocytic cells within the mesoderm of this mutant.

Effect of the Steel gene product on melanogenesis in avian neural crest cell cultures.

Mutations at the Steel (Sl) and dominant white spotting (W) loci affect three embryonic lineages: primordial germ cells, hemopoietic stem cells and neural-crest-derived melanocytes. The gene products of these loci are a peptide growth factor, called here stem cell factor (SCF), and its tyrosine kinase receptor, the proto-oncogene c-kit. We have studied how chicken recombinant SCF affects the development of melanocytes from quail neural crest cells in secondary culture under defined conditions. We observed that the total number of neural crest cells, of melanocytes and of their precursors was higher in the presence than in the absence of SCF. Labelling with bromodeoxyuridine showed that SCF had a modest and transient mitogenic effect on the neural crest population. SCF also enhanced the differentiation rate of melanocyte precursors, recognized by the "melanocyte early marker" monoclonal antibody (MelEM MAb), and of melanocytes, since the proportion of both subpopulations significantly increased in the presence of SCF. Finally, SCF increased the survival of the neural crest population since in its presence the total number of cells remained stable while it gradually declined in control cultures. Our results support the notion that SCF sustains the survival of the neural crest population and stimulates the rate of the melanogenic differentiation process.