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.     

Effect of thiostrepton and 3′-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis

The effect of the antibiotics thiostrepton and micrococcin on EF-T_u-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3′-terminus of aminoacyl-tRNA)(System I) or by methanol (‘uncoupled GTPase’, System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-T_u·GTP·70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the K^GTP_m is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases V^GTP_max, whereas K^GTP_m remains unaffected. Micrococcin is without any effect in both systems. The ‘uncoupled GTPase’ (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-T_u site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-T_u for GTP.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

An Ecteola-cellulose chromatography assay for 3′-phosphoadenosine 5′-phosphosulfate: Phenol sulfotransferase

A new assay procedure for phenol sulfotransferase which employs [35S]-3'-phosphoadenosine-5'-phosphosulfate as a sulfate donor and a variety of phenols as sulfate acceptors was developed. The appearance of the 35S-sulfated products or the disappearance of the [35S]-3'-phosphoadenosine-5'-phosphosulfate are determined simultaneously by chromatography of the assay incubation mixtures on Ecteola-cellulose columns, eluting with an NH4HCO3 step gradient. Various acidic, neutral, and basic phenols can be employed as substrates for phenol sulfotransferase using this procedure.     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

Reversible inactivation of soluble liver guanylate cyclase by disulfides

A new amino acid was isolated from the cuticle collagen of $\text{Ascaris lumbricoides}$ and characterized by ultraviolet, mass and nmr spectroscopies and chemical degradation. The results indicate that the compound is an isomer of trityrosine, having an ether linkage. The name “isotrityrosine” is proposed. Its structure suggests that it serves as a crosslink and plays a role in the organization of the collagen structure.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Thermographic visualization of cell death in tobacco and Arabidopsis

Pending cell death was visualized by thermographic imaging in bacterio‐opsin transgenic tobacco plants. Cell death in these plants was characterized by a complex lesion phenotype. Isolated cell death lesions were preceded by a colocalized thermal effect, as previously observed at sites infected by tobacco mosaic virus (TMV) ( Chaerle et al. 1999 Nature Biotechnology 17, 813–816). However, in most cases, a coherent front of higher temperature, trailed by cell death, initiated at the leaf base and expanded over the leaf lamina. In contrast to the homogenous thermal front, cell death was first visible close to the veins, and subsequently appeared as discrete spots on the interveinal tissue, as cell death spread along the veins. Regions with visible cell death had a lower temperature because of water evaporation from damaged cells. In analogy with previous observations on the localized tobacco–TMV interaction ( Chaerle et al. 1999 ), the kinetics of thermographic and continuous gas exchange measurements indicated that stomatal closure preceded tissue collapse. Localized spontaneous cell death could also be presymptomatically visualized in the Arabidopsis lsd2 mutant.     

Folic acid reverses nitric oxide synthase uncoupling and prevents cardiac dysfunction in insulin resistance: Role of Ca2+/calmodulin-activated protein kinase II

Nitric oxide synthase (NOS) may be uncoupled to produce superoxide rather than nitric oxide (NO) under pathological conditions such as diabetes mellitus and insulin resistance, leading to cardiac contractile anomalies. Nonetheless, the role of NOS uncoupling in insulin resistance-induced cardiac dysfunction remains elusive. Given that folic acid may produce beneficial effects for cardiac insufficiency partially through its NOS recoupling capacity, this study was designed to evaluate the effect of folic acid on insulin resistance-induced cardiac contractile dysfunction in a sucrose-induced insulin resistance model. Mice were fed a sucrose or starch diet for 8 weeks before administration of folic acid in drinking water for an additional 4 weeks. Cardiomyocyte contractile and Ca²⁺ transient properties were evaluated and myocardial function was assessed using echocardiography. Our results revealed whole body insulin resistance after sucrose feeding associated with diminished NO production, elevated peroxynitrite (ONOO⁻) levels, and impaired echocardiographic and cardiomyocyte function along with a leaky ryanodine receptor (RYR) and intracellular Ca²⁺ handling derangement. Western blot analysis showed that insulin resistance significantly promoted Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, which might be responsible for the leaky RYR and cardiac mechanical dysfunction. NOS recoupling using folic acid reversed insulin resistance-induced changes in NO and ONOO⁻, CaMKII phosphorylation, and cardiac mechanical abnormalities. Taken together, these data demonstrated that treatment with folic acid may reverse cardiac contractile and intracellular Ca²⁺ anomalies through ablation of CaMKII phosphorylation and RYR Ca²⁺ leak.     

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.