Saccharomyces cerevisiae Rot1 is an essential molecular chaperone in the endoplasmic reticulum

Molecular chaperones prevent aggregation of denatured proteins in vitro and are thought to support folding of diverse proteins in vivo. Chaperones may have some selectivity for their substrate proteins, but knowledge of particular in vivo substrates is still poor. We here show that yeast Rot1, an essential, type-I ER membrane protein functions as a chaperone. Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation. In vivo, the rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels. Furthermore, we demonstrate a physical and probably transient interaction of Rot1 with four of these proteins. Collectively, these results indicate that Rot1 functions as a chaperone in vivo supporting the folding of those proteins. Their folding also requires BiP, and one of these proteins was simultaneously associated with both Rot1 and BiP, suggesting that they can cooperate to facilitate protein folding. The Rot1-dependent proteins include a soluble, type I and II, and polytopic membrane proteins, and they do not share structural similarities. In addition, their dependency on Rot1 appeared different. We therefore propose that Rot1 is a general chaperone with some substrate specificity.     

Synergy of aldosterone and high salt induces vascular smooth muscle hypertrophy through up-regulation of NOX1

Aldosterone and excessive salt intake are obviously implicated in human arteriosclerosis. Aldosterone activates NADPH oxidase that induces superoxide production and cardiovascular cell hypertrophy. The activity of NADPH oxidase is influenced by the expression of its subunit, through which, vasoactive agents activate in the enzyme. Here, we show that aldosterone elicited overexpression of the NOX1 catalytic subunit of NADPH oxidase in the presence of high salt in A7r5 vascular smooth muscle cells. We also showed that NOX1 is a key subunit involved in physiological aldosterone-induced NADPH oxidase activation. Aldosterone dose-dependently increased NOX1 expression and NADPH activity, which subsequently caused superoxide over-production and A7r5 cell hypertrophy. However, aldosterone had little effect on any of NOX1, superoxide over-production and cell hypertrophy in NOX1 knock-down A7r5 cells. These results suggest that the aldosterone-induced effects are mainly generated through NOX1. Aldosterone-induced NOX1 over-expression was augmented by 145 mM sodium chloride, as compared with control medium containing 135 mM NaCl. However, NOX1 over-expression was not induced in the absence of aldosterone, even in the presence of 185 mM NaCl. The mineralocorticoid receptor antagonist, eplerenone, completely abolished NOX1 over-expression, indicating that aldosterone is essential for this process.     

Differential regulation of HSP70 expression by the JNK kinases SEK1 and MKK7 in mouse embryonic stem cells treated with cadmium

JNK, a member of the mitogen-activated protein kinases (MAPKs), is activated by the MAPK kinases SEK1 and MKK7 in response to environmental stresses. In the present study, the effects of CdCl2 treatment on MAPK phosphorylation and HSP70 expression were examined in mouse embryonic stem (ES) cells lacking the sek1 gene, the mkk7 gene, or both. Following CdCl2 exposure, the phosphorylation of JNK, p38, and ERK was suppressed in sek1-/- mkk7-/- cells. When sek1-/- or mkk7-/- cells were treated with CdCl2, JNK phosphorylation, but not the phosphorylation of either p38 or ERK, was markedly reduced, while a weak reduction in p38 phosphorylation was observed in sek1-/- cells. Thus, both SEK1 and MKK7 are required for JNK phosphorylation, whereas their role in p38 and ERK phosphorylation could overlap with that of another kinase. We also observed that CdCl2-induced HSP70 expression was abolished in sek1-/- mkk7-/- cells, was reduced in sek1-/- cells, and was enhanced in mkk7-/- cells. Similarly, the phosphorylation of heat shock factor 1 (HSF1) was decreased in sek1-/- mkk7-/- and sek1-/- cells, but was increased in mkk7-/- cells. Transfection with siRNA specific for JNK1, JNK2, p38, ERK1, or ERK2 suppressed CdCl2-induced HSP70 expression. In contrast, silencing of p38 or p38 resulted in further accumulation of HSP70 protein. These results suggest that HSP70 expression is up-regulated by SEK1 and down-regulated by MKK7 through distinct MAPK isoforms in mouse ES cells treated with CdCl2.     

The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Mutants of Corynebacterium glutamicum that were unable to grow under mild alkaline pH conditions were isolated by mutagenesis. Strain AL-43 exhibiting the highest sensitivity to alkaline pH among the mutants was selected and used to clone a DNA fragment that could complement the phenotype. Sequencing and subcloning of the cloned 4.0-kb EcoRI DNA fragment showed that the Cgl1281 gene was responsible for the complementation. The deduced amino acid sequence of Cgl1281 was found to show significant sequence similarity with CzcD, a Me²⁺/H⁺(K⁺) antiporter, from Bacillus subtilis and also possess the features of the cation diffusion facilitator (CDF) family: the presence of 6 putative transmembrane segments and a signature sequence, indicating that the gene product is a member of the CDF family. Chromosomal disruption of the Cgl1281 rendered C. glutamicum cells sensitive to alkaline pH as well as cobalt, while expression of the gene from a plasmid restored alkali-tolerance to the wild-type level and also led to increased cobalt resistance. These results demonstrated that the putative transporter of the CDF family mediates resistance to cobalt and also plays a physiological role in alkaline pH tolerance in C. glutamicum.     

Morphological analysis on the distribution of membrane lipids and a membrane protein,NAP-22, during neuronal development in vitro

Specific localization of membrane proteins based on the interactions with membrane lipids at various microdomains (MDs) is under active investigation, since the elucidation of the molecular mechanism of the interactions could reveal a novel concept of cell organization. Due to the strong interactions not only between lipids but also between lipids and proteins, these MDs are considered to be recovered in a detergent-resistant low-density membrane fraction (DRM) after detergent extraction and density-gradient centrifugation. Neurons take well-developed membrane systems during maturation and specific localization of various membrane components, not only proteins but also lipids, is essential for the establishment of the nervous system. In previous studies, we showed that NAP-22 is a major protein of neuronal DRM and binds liposomes in a cholesterol-dependent manner. In this study, we analyzed the localization of membrane lipids during neuronal maturation in vitro and compared their distribution with that of NAP-22. In an attempt to detect DRM-associated lipids, we observed the staining patterns of neurons treated with Triton-X-100 at 4 degrees C before fixation. Our results showed the less staining patterns of cholesterol and sphingomyelin at the axonal tips and a different staining pattern of two gangliosides, GM(1) and GD(3). The enrichment of cholesterol at the NAP-22 localizing spots was observed after the treatment of the detergent. Since the application of maitotoxin, a calcium ion channel, caused the diminution of NAP-22 and cholesterol positive spots, the distribution of these molecules are considered under the calcium regulation.     

Synthesis enables identification of the cellular target of leucascandrolide A and neopeltolide

Leucascandrolide A and neopeltolide are structurally homologous marine natural products that elicit potent antiproliferative profiles in mammalian cells and yeast. The scarcity of naturally available material has been a significant barrier to their biochemical and pharmacological evaluation. We developed practical synthetic access to this class of natural products that enabled the determination of their mechanism of action. We demonstrated effective cellular growth inhibition in yeast, which was substantially enhanced by substituting glucose with galactose or glycerol. These results, along with genetic analysis of determinants of drug sensitivity, suggested that leucascandrolide A and neopeltolide may inhibit mitochondrial ATP synthesis. Evaluation of the activity of the four mitochondrial electron transport chain complexes in yeast and mammalian cells revealed cytochrome bc(1) complex as the principal cellular target. This result provided the molecular basis for the potent antiproliferative activity of this class of marine macrolides, thus identifying them as new biochemical tools for investigation of eukaryotic energy metabolism.     

A combination of octanoate and oleate promotes in vitro differentiation of porcine intramuscular adipocytes

To understand the relationship between intramuscular adipogenesis in the pig and the supply fatty acids, we established a clonal porcine intramuscular preadipocyte (PIP) line from the marbling muscle tissue of female Duroc pig. Confluent PIP cells exhibited a fibroblastic appearance. Their adipogenic ability was investigated using confluent PIP cells after exchanging growth medium for adipogenic medium containing 50 ng/mL insulin, 0.25 microM dexamethasone, 2 mM octanoate, and 200 microM oleate. Appropriate concentrations of octanoate and oleate for the induction of adipogenesis were determined from the ability of cells to accumulate lipid and the toxicity of fatty acids. When cells were cultured in differentiation medium for 8 days, large numbers of lipid droplets were observed in differentiated PIP cells, and their cytosolic TG content increased in a time-dependent manner. While oleate only induced the expression of PPARgamma mRNA, but not that of C/EBPalpha, octanoate significantly induced the expression of both PPARgamma and C/EBPalpha mRNA. Octanoate and oleate accelerated the inducing effect of insulin and dexamethasone on the expression of aP2 mRNA. These results indicate that a combination of octanoate and oleate synergistically induced PIP adipogenesis, and that the stimulation of octanoate was essential to the trigger for the adipogenesis in PIP cells.     

Bioactive monoterpene glycosides conjugated with gallic acid from the leaves of Eucalyptus globulus

Two monoterpene glycosides, conjugated with gallic acid [globulusin A (1) and B (2)], together with four known compounds, cypellocarpin A (3), eucaglobulin (4), cuniloside (5) and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole beta-d-glucopyranoside (6), were isolated from hot-water extracts of the leaves of Eucalyptus globulus. The structures of compounds 1 and 2 were determined by 1D, 2D NMR and MS spectroscopic analyses. The absolute stereochemistry of 1 was determined by correlating the spectroscopic data with those of synthetic compound 6 with a known configuration. Globulusin A (1) and B (2), cypellocarpin A (3) and eucaglobulin (4), scavenged DPPH free radicals and globulusin A (1) showed a higher antioxidant activity than the other tested compounds, with an IC50 of 3.8microM. Globulusin A (1) and eucaglobulin (4) concentration-dependently suppressed inflammatory cytokine production, tumor-necrosis factor-alpha and interleukin-1beta in cultured human myeloma THP-1 cells co-stimulated with phorbol myristate acetate. These compounds also inhibited melanogenesis in cultured murine melanoma B16F1 cells, without any significant cytotoxicity. These results suggested that globulusin A (1) and eucaglobulin (4), which were isolated as antioxidants from E. globulus, also had anti-inflammatory as well as anti-melanogenesis activity.     

The versatility of Shigella effectors

When Shigella infect the intestinal epithelium, they deliver several effectors through the type III secretion system (T3SS) into the surrounding space and directly into the host-cell cytoplasm, where they can mimic and usurp host cellular functions or subvert host-cell signalling pathways and the immune response. Although bacterial strategies and mechanisms of infection vary greatly, recent studies of Shigella effectors have revealed that Shigella possess a highly evolved strategy for infection.