Proteomic analysis of responses to osmotic stress in laboratory and sake-brewing strains of Saccharomyces cerevisiae

The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics.     

Kei1: A Novel Subunit of Inositolphosphorylceramide Synthase,Essential for Its Enzyme Activity and Golgi Localization

Fungal sphingolipids have inositol-phosphate head groups, which are essential for the viability of cells. These head groups are added by inositol phosphorylceramide (IPC) synthase, and AUR1 has been thought to encode this enzyme. Here, we show that an essential protein encoded by KEI1 is a novel subunit of IPC synthase of Saccharomyces cerevisiae. We find that Kei1 is localized in the medial-Golgi and that Kei1 is cleaved by Kex2, a late Golgi processing endopeptidase; therefore, it recycles between the medial- and late Golgi compartments. The growth defect of kei1-1, a temperature-sensitive mutant, is effectively suppressed by the overexpression of AUR1, and Aur1 and Kei1 proteins form a complex in vivo. The kei1-1 mutant is hypersensitive to aureobasidin A, a specific inhibitor of IPC synthesis, and the IPC synthase activity in the mutant membranes is thermolabile. A part of Aur1 is missorted to the vacuole in kei1-1 cells. We show that the amino acid substitution in kei1-1 causes release of Kei1 during immunoprecipitation of Aur1 and that Aur1 without Kei1 has hardly detectable IPC synthase activity. From these results, we conclude that Kei1 is essential for both the activity and the Golgi localization of IPC synthase.     

Occurrence of spermic diploid and aspermic triploid forms of Fasciola in Vietnam and their molecular characterization based on nuclear and mitochondrial DNA

Fasciola spp. found in Asian countries are diversified in nature, and they should therefore be characterized by spermatogenesis, ploidy and genetic differentiation as well as morphology. The present study showed that spermic diploid and aspermic triploid forms of Fasciola occurred in Vietnam. The spermic diploid specimens were accurately identified as F. gigantica, while the aspermic triploids could not be identified on the basis of their morphology by the ratio of body length and width and DNA sequences of nuclear ribosomal ITS1 and mitochondrial NDI and COI genes. The molecular data also indicated that Vietnamese aspermic triploids might be hybrids and/or their offspring between Fasciola hepatica and F. gigantica, because they showed the ITS1-Fh/Fg haplotype, which had chimeric sequences of the two species. Furthermore, the aspermic triploids seem to have originated in countries other than Vietnam and to have rapidly spread to that country with infected animals.     

Design, synthesis, and evaluation of cyclic amide/imide-bearing hydroxamic acid derivatives as class-selective histone deacetylase (HDAC) inhibitors

A series of hydroxamic acid derivatives bearing a cyclic amide/imide group as a linker and/or cap structure, prepared during our structural development studies based on thalidomide, showed class-selective potent histone deacetylase (HDAC)-inhibitory activity. Structure-activity relationship studies indicated that the steric character of the substituent introduced at the cyclic amide/imide nitrogen atom, the presence of the amide/imide carbonyl group, the hydroxamic acid structure, the shape of the linking group, and the distance between the zinc-binding hydroxamic acid group and the cap structure are all important for HDAC-inhibitory activity and class selectivity. A representative compound (30w) showed potent p21 promoter activity, comparable with that of trichostatin A (TSA), and its cytostatic activity against cells of the human prostate cell line LNCaP was more potent than that of the well-known HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA).     

Cellular signaling of Dock family proteins in neural function

Dock180-related proteins are genetically conserved from Drosophila and C. elegans to mammals and are atypical types of guanine–nucleotide exchange factors (GEFs) for Rac and/or Cdc42 of small GTPases of the Rho family. Eleven members of the family occur in mammalian cells, each playing key roles in many aspects of essential cellular functions such as regulation of cytoskeletal organization, phagocytosis, cell migration, polarity formation, and differentiation. This review will summarize the newly accumulated findings concerning the Dock180-related proteins' molecular and cellular functions, emphasizing the roles of these proteins in neuronal cells and glial cells as well as their interactions in the central and peripheral nervous systems.     

Clinical significance on MicroScan Rapid plus series using various antibiotic-resistant bacteria

MicroScan Rapid plus Neg II Series and MicroScan Rapid plus Pos Series by Siemens Healthcare Diagnostics K.K. are the panels which enable to measure identification and antimicrobial susceptibility testing quickly and we have confirmed that it is useful for detecting drug resistance bacteria. As the identification result of comparing Rapid plus series with the current panel by using 143 strains of various drug resistance bacteria, Gram positive cocci was 87. 7%, glucose fermenter was 100% and glucose non-fermenter was 77.3 in Gram negative bacilli. On the evaluation of antimicrobial susceptibility testing, Rapid plus series, in comparison with the current panel, confirmed the lower tendency of MIC value on some drugs, but it basically presented the good concordance rate. In terms of the reporting time of antimicrobial agent, non-fermenter or MRCNS reported the result as needed after 8 hours and it took a little longer time for the report of antimicrobial agent. On the other hand, 80% or higher of antimicrobial agent on panel was reported for intestinal bacteria in 4.5 hours and for MRSA in 6.5 hours. It enabled to report the testing result on the same day. Due to the results above, Rapid plus series was highly valued on the usability, such as the early detection of drug resistance bacteria and the selection of therapeutic agents.     

Identification of a royal jelly glycoprotein that carries unique complex-type N-glycans harboring the T-antigen (Galβ1-3GalNAc) unit

In this study, we identified a royal jelly glycoprotein (RJG) that carries a unique complex-type N-glycans harboring the T-antigen (Galβ1-3GalNAc) unit. The amino acid sequence of the tryptic glycopeptide harboring the T-antigen unit was G-E-S-L-X-K (X might be glycosylated Asn), confirmed in the major royal jelly glycoprotein 1 (MRJP1), which is also expressed in the mushroom body of the honeybee brain.     

Perlecan deficiency causes muscle hypertrophy,a decrease in myostatin expression,and changes in muscle fiber composition

Perlecan is a component of the basement membrane that surrounds skeletal muscle. The aim of the present study is to identify the role of perlecan in skeletal muscle hypertrophy and myostatin signaling, with and without mechanical stress, using a mouse model (Hspg2^?/?-Tg) deficient in skeletal muscle perlecan. We found that myosin heavy chain (MHC) type IIb fibers in the tibialis anterior (TA) muscle of Hspg2^?/?-Tg mice had a significantly increased fiber cross-sectional area (CSA) compared to control (WT-Tg) mice. Hspg2^?/?-Tg mice also had an increased number of type IIx fibers in the TA muscle. Myostatin and its type I receptor (ALK4) expression was substantially decreased in the Hspg2^?/?-Tg TA muscle. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the Hspg2^?/?-Tg muscle, suggesting that myostatin expression and its signaling were decreased in the Hspg2^?/?-Tg muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in Hspg2^?/?-Tg mice, we performed tenotomy of the plantaris (fast) muscle and the soleus (slow) muscle. Mechanical overload on the plantaris muscle of Hspg2^?/?-Tg mice significantly increased wet weights compared to those of control mice, and unloaded plantaris muscles of Hspg2^?/?-Tg mice caused less decrease in wet weights compared to those of control mice. The decrease in myostatin expression was significantly profound in the overloaded plantaris muscle of Hspg2^?/?-Tg mice, compared with that of control mice. In contrast, overloading the soleus muscle caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition, and for regulating myostatin signaling.     

Bladder dysfunction after acute urinary retention in the rats: a novel over active bladder model

As there is increasing evidence that benign prostatic hyperplasia and its related acute urinary retention (AUR) induce over active bladder (OAB) syndrome, we investigated the effects of AUR on bladder function over a 4-week period in a rat model. Ten-week-old female Sprague-Dawley rats were used in this study. AUR was induced by clamping the distal urethra of each rat with a small clip, and then infusing 3 ml (0.6 ml/min) of saline with an infusion pump through a transurethral catheter (22G). The obstruction was sustained for 60 min and the clip was removed and then the bladder was allowed to drain through the catheter. The bladder function was estimated by voiding behavior studies (at 3 days, 1, 2, 3, and 4 weeks), cystometric studies (at 2 and 4 weeks) and organ bath studies using KCl and carbachol (at 2 and 4 weeks). Furthermore, we evaluated histological changes in the rat bladder 2 and 4 weeks after the induction of AUR. The same parameters were also measured in non-AUR rats (control group). The rat bladder weight in the AUR group at 2 weeks was significantly larger than that of the controls, and returned to the control level 4 weeks after the AUR episode. The voiding behavior studies showed significant increase in micturition frequency per day and decrease in single voiding volume 3 days after the induction of AUR, and this voiding behavior was continued for more than 2 weeks. The cystometric studies showed a significant decrease in single-voided volume at 2 weeks rat. However, no significant changes of the other parameters were observed in the rats. The histological studies showed significant infiltration of neutrophils and lymphocytes, as well as increase in turnover of epithelium in AUR rats at 2 weeks, while significant increases in fibrosis in submucosal layer were observed in AUR rats at 4 weeks. This study demonstrated that bladder dysfunction in the rat model caused by AUR needs more than 2 weeks of recovery period. The AUR-associated alterations in the bladder may represent a key clue to understand the underlying pathophysiological mechanisms, which take place in OAB syndrome.