Occurrence of N-methyl- -aspartate in bivalves and its distribution compared with that of N-methyl- -aspartate and , -aspartate

The presence of N-methyl- -aspartate (NMLA) was demonstrated in bivalves, Corbicula sandai and Tapes japonica. To our knowledge, this is the first report on the occurrence of NMLA in animal tissues. NMLA in bivalve tissues was identified according to the following findings; (a) its derivatives with (+)- and (−)- 1-(9-fluorenyl)ethyl chloroformate (FLEC) behaved identically with those of authentic NMLA, respectively, on high-performance liquid chromatography (HPLC), (b) its derivatives with (+)- and (−)- FLEC behaved identically with (−)- and (+)-FLEC derivatives of authentic N-methyl- -aspartate (NMDA), respectively, on HPLC and (c) its behavior on thin-layer chromatography was the same as those of authentic NMLA. We also describe the distribution of NMDA, and - and -aspartate, to which N-methylaspartate enantiomers are structurally related. NMDA was more widely dirtributed than NMLA in bivalves. These bivalves containing NMLA showed lower -aspartate contents and /( + ) ratios of aspartate, than the bivalves containing NMDA.     

GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.     

β-Cryptoxanthin Alleviates Diet-Induced Nonalcoholic Steatohepatitis by Suppressing Inflammatory Gene Expression in Mice

Recent nutritional epidemiological surveys showed that serum β-cryptoxanthin inversely associates with the risks for insulin resistance and liver dysfunction. Consumption of β-cryptoxanthin possibly prevents nonalcoholic steatohepatitis (NASH), which is suggested to be caused by insulin resistance and oxidative stress from nonalcoholic fatty liver disease. To evaluate the effect of β-cryptoxanthin on diet-induced NASH, we fed a high-cholesterol and high-fat diet (CL diet) with or without 0.003% β-cryptoxanthin to C56BL/6J mice for 12 weeks. After feeding, β-cryptoxanthin attenuated fat accumulation, increases in Kupffer and activated stellate cells, and fibrosis in CL diet-induced NASH in the mice. Comprehensive gene expression analysis showed that although β-cryptoxanthin histochemically reduced steatosis, it was more effective in inhibiting inflammatory gene expression change in NASH. β-Cryptoxanthin reduced the alteration of expression of genes associated with cell death, inflammatory responses, infiltration and activation of macrophages and other leukocytes, quantity of T cells, and free radical scavenging. However, it showed little effect on the expression of genes related to cholesterol and other lipid metabolism. The expression of markers of M1 and M2 macrophages, T helper cells, and cytotoxic T cells was significantly induced in NASH and reduced by β-cryptoxanthin. β-Cryptoxanthin suppressed the expression of lipopolysaccharide (LPS)-inducible and/or TNFα-inducible genes in NASH. Increased levels of the oxidative stress marker thiobarbituric acid reactive substances (TBARS) were reduced by β-cryptoxanthin in NASH. Thus, β-cryptoxanthin suppresses inflammation and the resulting fibrosis probably by primarily suppressing the increase and activation of macrophages and other immune cells. Reducing oxidative stress is likely to be a major mechanism of inflammation and injury suppression in the livers of mice with NASH.     

Relationship between progesterone and estradiol contents of follicular fluid and the morphological appearance of granulosa cells of follicles coexisting with corpora lutea in bovine ovaries

Bovine ovaries (n=149) bearing follicles (>5 mm) coexisting with mature corpora lutea (CL;>10 mm) were obtained at a local abattoir without regard for the reproductive status of the donor cows. Most corpora lutea were 21 to 25 mm in diameter, and nearly half of the largest follicles were 11 to 15 mm in diameter. When oocytes were aspirated from follicles 16 to 30 mm in diameter, approximately 60% of them proved to be degenerated. Concentrations of progesterone (P4) and estradiol-17beta (E2) in the follicular fluid of 23 follicles (>10 mm) were determined. Progesterone and estradiol-17beta were found to be the major hormone in 16 (69.6%) and 7 (30.4%) of the follicles, respectively. Light-microscope observations of the granulosa cells of the same 23 follicles showed that 7 were deficient in mural granulosa cells, and that 15 of the remaining 16 follicles were atretic or luteinizing. Ultrastructural observations of granulosa cells revealed many lipid droplets in the cytoplasm of follicles coexisting with mature CL, suggesting the initiation of luteinization. These results show that approximately 70% of the follicles were P4-dominant and that more than 95% of them were morphologically degenerated. Thus it is suggested that morphological signs of atresia precede changes in the concentrations of hormones in the follicular fluid of follicles coexisting with corpora lutea (>10 mm) during the middle of the estrous cycle.     

Vitamin K2 Biosynthetic Enzyme,UBIAD1 Is Essential for Embryonic Development of Mice

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K₂ biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1 ^−/−) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1 ^−/− embryonic stem (ES) cells failed to synthesize vitamin K₂ but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1 ^+/− mice developed normally, exhibiting normal growth and fertility. Vitamin K₂ tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K₂ synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1 ^+/− E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1 ^−/− mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1 ^+/− mice. These results suggest that UBIAD1 is responsible for vitamin K₂ synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K₂, but may have additional functions beyond the biosynthesis of vitamin K₂.     

The rat mast cell antigen AD1 (homologue to human CD63 or melanoma antigen ME491) is expressed in other cells in culture.

Previously we reported that the mAb AD1 recognized a heavily glycosylated 50- to 60-kDa protein (AD1 Ag) sterically close to the high-affinity IgE receptor on rat basophilic leukemia (RBL-2H3) cells. The N-terminal amino acid sequence of the AD1 Ag was nearly identical to that of human CD63 (melanoma-associated Ag ME491). In this study we cloned the cDNA of AD1 Ag from a rat basophilic leukemia 2H3 cDNA library. An open reading frame of 238 amino acids was identified that contained the N-terminal 43 amino acid sequence. No evidence of a signal peptide was found. However, four predominantly hydrophobic stretches of sequence were predicted to form membrane-spanning helices, and three putative N-glycosylation sites were identified. The AD1 Ag and CD63 were highly conserved between rat and human, suggesting that the sequence of this protein is important for its function. By immunostaining various rat tissues, the AD1 Ag was found localized to mast cells. However, it was located to lysosomes, secretory granules and the plasma membrane of RBL-2H3 cells and to lysosomes and plasma membrane of many other cultured cell lines. The AD1 Ag could be induced by placing cells in culture. Fibroblasts and hepatocytes freshly isolated from rat embryos stained very weakly for AD1 Ag; however, after 24 to 48 h in culture they were strongly positive. This increase in the expression of the AD1 Ag was accompanied by an increase in detectable RNA message. Therefore, AD1/ME491/CD63 Ag is a mast cell marker in tissue, but is also associated with other cells in culture.     

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.