A newly established mutant strain with mild-type ocular coloboma (retinochoroidal coloboma without microphthalmia) in albino mice

BACKGROUND: A complicated malformation of the fundus accompanied by typical ocular coloboma was detected in albino fatty liver Shionogi (FLS) mice. We elucidated a new type of 3-dimensional anomalous structure inside the eye in this mouse strain. METHODS: The fundi of FLS mice aged 1, 3, 5, and 20 weeks were observed intensively, both macroscopically and by light microscopy. For the prenatal study, coronal serial sections of eyes of FLS embryos were examined by light microscopy on gestation day (GD) 15.0. RESULTS: The frequency of ocular coloboma was almost 70% in FLS mice, and the inheritance mode of this anomaly is suggested to be autosomal recessive with incomplete penetrance. Stereoscopic observation and light microscopy revealed that the mice had characteristic fundus features at any age during the postnatal period. Following ectopic ciliary epithelia, the surface of the retina protruded like a roof, and on the opposite side of the "roof," a translucent membrane without retinal tissue and choroidal tissue was also consistently detected in the inferior part of the fundus. On GD 15.0, the inner layer and the outer layer were not normally fused at the optic fissure, where a part of the outer layer was absent and the irregular fold of the inner layer was conspicuous in the colobomatous eye of the FLS embryo. CONCLUSIONS: The characteristics of the ocular coloboma in FLS mice are thought to be similar to a mild-type malformation in humans. These ocular defects seem to be situated along the failed fetal optic fissure.     

Chicken ornithine transcarbamylase: purification and some properties

Ornithine transcarbamylase [EC 2.1.3.3] has been purified from chick kidney to homogeneity. The molecular weight is 110,000 as determined by gel filtration. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the enzyme showed that the enzyme exists as a trimer of identical subunits of 36,000 daltons like other mammalian species ornithine transcarbamylases. In 0.1 M triethanolamine/HCl, the apparent optimum pH of the purified enzyme was 7.5 in the presence of 5 mM ornithine. The curve shifted toward a more alkaline region with a decrease in ornithine concentration. The specific activity of the purified enzyme as 77 units at pH 7.5. The Km for carbamyl phosphate was 0.11 mM and the Km for ornithine was 1.21 mM. With an increase in pH, a decrease in Km values for ornithine and an increase in the extent of inhibition by ornithine were observed. On using antibody against bovine liver ornithine transcarbamylase, the precipitin lines for the chick and bovine enzymes showed a spur pattern. Even when excess amounts of the antibody were added, the chick enzyme did not lose the activity while the bovine enzyme activity was inhibited completely.     

Does Enterohemorrhagic Escherichia coli O157:H7 Enter the Viable but Nonculturable State in Salted Salmon Roe?

An outbreak caused by salted salmon roe contaminated with enterohemorrhagic Escherichia coli O157 occurred in Japan in 1998. Since about 0.75 to 1.5 viable cells were estimated to cause infection, we presumed that O157 might enter the viable but nonculturable (VNC) state in salted salmon roe and consequently that viable cell numbers might be underestimated. Although patient-originating O157 cells could not grow on agar plates after 72 h of incubation in 13% NaCl, they were resuscitated in yeast extract broth, and more than 90% of the cells were shown to be viable by fluorescent staining, suggesting that almost all of them could enter the VNC state in NaCl water. Roe-originating O157 was resistant to NaCl because it could grow on agar after 72 h of incubation in NaCl water, but about 20% of cells appeared to enter the VNC state. Therefore, germfree mice were infected with O157 to examine the resuscitation of cells in the VNC state and the retention of pathogenicity. O157 that originated in roe, but not patients, killed mice and was isolated from the intestine. However, these isolates had become sensitive to NaCl. O157 cells of roe origin incubated in normal media also killed mice and were isolated from the intestine, but they also became transiently NaCl sensitive. We therefore propose that bacterial cells might enter the VNC state under conditions of stress, such as those encountered in vivo or in high salt concentrations, and then revive when those conditions have eased. If so, the VNC state in food is potentially dangerous from a public health viewpoint and may have to be considered at the time of food inspection. Finally, the establishment of a simple recovery system for VNC cells should be established.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP⁺-binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP⁺ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP⁺ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PD^def). We first solved the crystal structure for G6PD^K403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP⁺ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PD^def), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PD^K403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PD^def and provide a foundation for future drug discovery efforts.     

Enhancement of Protease Activity in Transesterification of Glycidol with Vinyl n-butyrate by Entrapment into Alkyl-substituted Silicates and Pretreatment with a Substrate

Proteases originating from Aspergillus melleus (Protease P) and Bacillus subtillis (Prolether FG-F) were entrapped into organic-inorganic hybrid silicates on Celite 545 by the sol-gel method, and their activities measured at 35°C for transesterification of chiral glycidol with vinyl n-butyrate in isooctane. n-Butyl- and dimethyl-substituted silicates provided 12.6 times higher activities with Protease P and 5.5 times with Prolether FG-F, respectively, than those deposited on Celite 545. Although pretreatment of those immobilized proteases with the chiral glycidol affected transesterification activities of both enantiomers, the ratio of the initial transesterification rate of (S)-(?)-glycidol to that of (R)-(+)-glycidol, remained unchanged.     

Japanese Rice Wine can reduce psychophysical stress-induced depression-like behaviors and Fos expression in the trigeminal subnucleus caudalis evoked by masseter muscle injury in the rats

We determined if Japanese Rice Wine (Sake) had inhibitory effects on stress-induced enhancement of masseter muscle (MM) nociception in the rats. Male rats were subjected to the repeated forced swim stress (FS) or sham conditionings from Day ?3 to ?1. Daily administration of Sake or saline was conducted after each stress conditioning. At Day 0 the number of Fos positive cells, a marker for neural activity, was quantified at the trigeminal subnucleus caudalis (Vc) region by MM injury with formalin. FS increased MM-evoked Fos expression in the Vc region, which was inhibited by Sake compared to saline administration. Sake did not alter the number of Fos positive cells under sham conditions, indicating that inhibitory roles of Sake on neural activity in the Vc region were seen under FS conditions. These findings indicated that Sake had inhibitory roles on stress-induced MM nociception at the Vc region in our experimental conditions.     

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress

Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.     

Accumulation of Uridinediphospho-N-acetylmuramyl-peptides Induced by Glycine and Penicillin

The genes POX2 and POX4, which encode the subunits (PXP-2 and PXP-4) of peroxisomal fatty acyl-coenzyme A oxidase of Candida tropicalis, were introduced into the related yeast Candida maltosa. The cells transformed with POX2 or POX4 gave much PXP-2 or PXP-4 in the purified peroxisomes. The polypeptides associated with the heterologous organelle were resistant to added protease, implying that they were transported into the peroxisomes. Genes for curtailed versions of PXP-4 were constructed in vitro and introduced into the host cells. Peptide-C, the COOH-terminal two-thirds of PXP-4, was efficiently transported into the host peroxisomes, and the polypeptide containing the NH₂-terminal one-third was also, in much lesser amount. These and other results suggested that there were at least two regions of peroxisomal targeting information in PXP-4 and the primary information was internal. The deletions in Peptide-C inhibited the transport of many, but not all, of the host-cell peroxisomal polypeptides. This suggested heterogeneous transport systems on the peroxisomal membrane.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.