A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.     

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI)

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.     

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

Key message

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

     

A Large Scale Analysis of Protein-Protein Interactions in the Nitrogen-fixing Bacterium Mesorhizobium loti

Global viewing of protein–protein interactions (PPIs)is a useful way to assign biological roles to large numbersof proteins predicted by complete genome sequence. Here, wesystematically analyzed PPIs in the nitrogen-fixing soil bacteriumMesorhizobium loti using a modified high-throughput yeast two-hybridsystem. The aims of this study are primarily on the providingfunctional clues to M. loti proteins that are relevant to symbioticnitrogen fixation and conserved in other rhizobium species,especially proteins with regulatory functions and unannotatedproteins. By the screening of 1542 genes as bait, 3121 independentinteractions involving 1804 proteins (24% of the total proteincoding genes) were identified and each interaction was evaluatedusing an interaction generality (IG) measure and the generalfeatures of the interacting partners. Most PPIs detected inthis study are novel interactions revealing potential functionalrelationships between genes for symbiotic nitrogen fixationand signal transduction. Furthermore, we have predicted theputative functions of unannotated proteins through their interactionswith known proteins. The results described here represent newinsight into protein network of M. loti and provide useful experimentalclues to elucidate the biological function of rhizobial genesthat can not be assigned directly from their genomic sequence.     

Thalidomide inhibits epidermal growth factor-induced cell growth in mouse and human monocytic leukemia cells via Ras inactivation

The effect of thalidomide on epidermal growth factor (EGF)-induced cell growth was examined. Thalidomide inhibited EGF-induced cell growth in mouse and human monocytic leukemia cells, RAW 264.7, U937 and THP-1. Thalidomide inhibited EGF-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2, but not p38 and stress-activated protein kinase (SAPK)/JNK. The phosphorylation of MEK1/2 and Raf at Ser 338 as the upstream molecules of ERK 1/2 was also prevented by thalidomide. Further, it inhibited EGF-induced Ras activation through preventing the transition to GTP-bound active Ras. Thalidomide inhibited the Ras activation induced by lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF) as well as EGF. There was no significant difference in the expression and function of EGF receptor between thalidomide-treated and non-treated cells. Therefore, thalidomide was suggested to inhibit EGF-induced cell growth via inactivation of Ras.     

Microsomal Electron Transfer in Higher Plants: Cloning and Heterologous Expression of NADH-Cytochrome b5 Reductase from Arabidopsis

AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b₅ reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b₅, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b₅ reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b₅ reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b₅ reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b₅ (AtB5-A), whereas no Cyt b₅ reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b₅ with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase can reduce Cyt b₅ and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.     

In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.     

Apoptosis in the aging process

Although many hypotheses have been proposed to explain the aging process, the exact mechanisms are not well defined. Recent accumulating evidence indicates that dysregulation of the apoptotic process may be involved in some aging processes; however, it is still debatable how exactly apoptosis is expressed during aging in vivo. In this review, we discuss recent findings related to apoptosis of individual organs during aging and their significance. We demonstrate that aging enhances apoptosis and susceptibility to apoptosis in several types of intact cells. In contrast, in certain genetically damaged, initiated, and preneoplastic cells, aging suppresses these age-associated apoptotic changes. In various cells, apoptosis enhances the elimination of damaged and dysfunctional cells presumably caused by oxidative stress, glycation, and DNA damage. In these cases, the incidence of apoptosis correlates with the level of accumulated injury. It is concluded that apoptosis plays an important role in the aging process and tumorigenesis in vivo probably as an inherent protective mechanism against age-associated tumorigenesis.     

Peri-operative immunotherapy with OK-432

Pre- and postoperative intradermal administration of OK-432 enhanced the SU-PS skin reaction in patients with gastric cancer, but failed to prevent a fall in the NK activity induced by the operation.The change in NK activity was not associated with a change in the proportion of Leu 7-positive cells, but was related to Leu 11a-positive cells. Intradermal injection of OK-432 increased the proportion of Leu 7-positive cells in the patients in whom they accounted for less than 20% of lymphocyte population. The case was the same with Leu 11a-positive cells.Intravenous injection of OK-432 tended to increase suppressor-inducer T cells (CD4⁺2HA⁺ cells), B cells and Leu 7-positive cells. Particularly, the proportions of OK-M₁-positive cells and MHC class II antigen-positive cells increased in all patients. Immunotherapy with OK-432 given intravenously at a dose of 0.1 KE appeared to be safe because no side effects were essentially observed.