Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Patterns and sources of variation in Daphnia phosphorus content in nature

It has recently been shown that Daphnia can vary in the phosphorus (P)-content of their body tissues, but the relative importance of genetic versus environmental causes for this variation is unexplored. We measured variation in P-content (as % body mass) of Daphnia from eight lakes and conducted experiments to contrast three sources of variation: interspecific variation, clonal variation and phenotypic plasticity. Daphnia P-content decreased with increasing seston C:P ratio across lakes. This relationship reflected both inter- and intraspecific variation. Daphnia parvula and D. dubia exhibited high P-content and were found in shallow lakes with low C:P seston, whereas D. pulicaria had low P-content and was found in deep, stratified lakes having high C:P seston. Populations of D. dentifera spanned this lake gradient and exhibited P-content that was negatively related to seston C:P. Evidence for phenotypic plasticity came from experiments with D. pulicaria and D. dentifera collected from a lake with P-deficient seston and fed a P-sufficient diet in the laboratory. In addition, populations of D. dentifera differed in P-content even after 7 d of feeding on P-sufficient resources, suggesting within-species clonal variation. However, mesocosm experiments revealed broad and surprisingly continuous variation in the P-content of individual clones of D. pulex (range 1.54–1.05%) and D. mendotae (1.51–1.07%) over a gradient in dietary C:P. The broad range in P-content exhibited by individual clones, acclimated for generations, suggests that variation in Daphnia P-content from laboratory experiments needs to be interpreted with caution. These results also show that phenotypic variation in response to environment can be a larger source of variation in P-content than genetic differences within or among species.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.     

Effect of algal inoculation on the yield and vitamin C content of two varieties of tomato

Summary Two varieties of tomato (Pusa Rubi and Selection 120) positively responded to algal inoculation in terms of the yield of fruits and shoots, but there was no significant effect on the vitamin C content of the fruits. A combined application of urea and algae was more effective than the application of urea alone.     

Microphytobenthic biomass assessment by pigment analysis: comparison of spectrophotometry and High Performance Liquid Chromatography methods

Samples of microphytobenthos from the Tagus estuary were analysed for photosynthetic pigments by spectrophotometry and High Performance Liquid Chromatography (HPLC). Chlorophyll a values obtained with HPLC and spectrophotometry methods presented a highly significant positive correlation for both spectrophotometric methods used (with and without the correction for pheopigments), but this relationship depended on the type of sediment. We concluded that spectrophotometric methods give reliable Chl-a values, being suited for routine analysis, when a vast number of replicates is needed. However, for the correct estimation of pheopigments, HPLC analysis is indispensable. In the literature, Chl-a estimations are expressed per content (μg g⁻¹) or concentration (mg m⁻²). We discuss the influence of sediment type on the results depending on the type of unit used, and propose a simple conversion factor based on sediment water content.     

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions

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