The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.     

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.     

Expression of Arabidopsis thaliana xylose isomerase gene and its effect on ethanol production in Flammulina velutipes

To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes.     

Establishment of laurel forest vegetation in adjacent secondary forest in central Japan

Distributions of lucidophyllous species are limited due to the fragmentation of laurel forest. On Komayama Hill in central Japan, we evaluated the colonization of typical lucidophyllous vascular plants from a 350-year-old laurel forest into adjacent abandoned secondary forest for conservation and restoration purposes. A total of 14 consecutive subplots were established along the vegetation border between the two forests (length, 30 m; width, 5 m), extending 70 m into the secondary forest; 18 quadrats of old-growth forest were surveyed. Edge effects of old-growth forest were found to play an important role in re-establishing lucidophyllous saplings and seedlings in the secondary forest. In particular, the abundances of the four dominant canopy species of the old-growth forest significantly decreased with increasing distance. Hence, they are expected to colonize further into the secondary forest and, ultimately, to dominate the canopy. However, the number of lucidophyllous species did not change with distance. Species such as Ficus nipponica, Damnacanthus indicus, Ilex integra, and Lemmaphyllum microphyllum were near-completely or completely limited to the old-growth forest. They are known to be negatively affected by forest fragmentation and were observed to be struggling to colonize the exterior of the old-growth forest even after 60 years of abandonment. Their absence highlighted the limited colonization capacities of some old-growth forest species and underlined the time required for habitat restoration following human disturbance. We conclude that it is important to consider the population dynamics of dominant canopy species and the colonization of these interior species when assessing the habitat expansion of lucidophyllous species and hence the restoration of degraded lands.     

Bifidobacteria Abundance-Featured Gut Microbiota Compositional Change in Patients with Behcet’s Disease

Gut microbiota compositional alteration may have an association with immune dysfunction in patients with Behcet’s disease (BD). We conducted a fecal metagenomic analysis of BD patients. We analyzed fecal microbiota obtained from 12 patients with BD and 12 normal individuals by sequencing of 16S ribosomal RNA gene. We compared the relative abundance of bacterial taxa. Direct comparison of the relative abundance of bacterial taxa demonstrated that the genera Bifidobacterium and Eggerthella increased significantly and the genera Megamonas and Prevotella decreased significantly in BD patients compared with normal individuals. A linear discriminant analysis of bacterial taxa showed that the phylum Actinobacteria, including Bifidobacterium, and the family Lactobacillaceae exhibited larger positive effect sizes than other bacteria in patients with BD. The phylum Firmicutes and the class Clostridia had large effect sizes in normal individuals. There was no significant difference in annotated species numbers (as numbers of operational taxonomic unit; OTU) and bacterial diversity of each sample (alpha diversity) between BD patients and normal individuals. We next assigned each sample to a position using three axes by principal coordinates analysis of the OTU table. The two groups had a significant distance as beta diversity in the 3-axis space. Fecal sIgA concentrations increased significantly in BD patients but did not correlate with any bacterial taxonomic abundance. These data suggest that the compositional changes of gut microbes may be one type of dysbiosis (unfavorable microbiota alteration) in patients with BD. The dysbiosis may have an association with the pathophysiology of BD.     

Carbenoxolone Disodium Treatment for Canine Pituitary-Dependent Hyperadrenocorticism

Pituitary-dependent hyperadrenocorticism (PDH) is mainly caused by pituitary corticotroph tumors in dogs. A characteristic feature of corticotroph tumors is their resistance to negative feedback by glucocorticoids. In some animal species, including dogs, the aberrant expression of 11β-hydroxysteroid dehydrogenase (11HSD), a cortisol metabolic enzyme, is observed in corticotroph tumors. We previously reported that carbenoxolone (CBX), an inhibitor of 11HSD, suppressed ACTH secretion from the pituitary gland, and decreased cortisol concentrations in healthy dogs. Therefore, the aim of this study was to investigate the therapeutic effects of CBX on dogs with PDH. Six dogs with PDH were treated with 60 to 80 mg/kg/day of CBX for 6 weeks, followed by trilostane, which is a commonly used agent for canine PDH. CBX treatment led to a gradual decrease in both basal and in corticotropic releasing hormone (CRH)-stimulated plasma ACTH concentrations and CRH-stimulated serum cortisol concentrations, without side effects. However, basal and stimulated ACTH and cortisol concentrations remained higher than those of healthy dogs, and clinical symptoms such as polydipsia and polyuria were not ameliorated. After a 2-week wash-out interval, trilostane was administered for 2 weeks. Although basal plasma ACTH concentrations were higher after trilostane treatment than CBX treatment, polydipsia and polyuria resolved in all six dogs. The reason for the lack of improvement in polydipsia and polyuria with CBX treatment is unclear. Other mechanisms, in addition to a partial decrease in ACTH secretion, are likely to be involved. In conclusion, this is the first study to report the in vivo effects of CBX in dogs with PDH. The findings suggest that CBX inhibits ACTH secretion from canine pituitary tumors, resulting in lower cortisol concentrations.     

Pathogenicity of Genetically Similar,H5N1 Highly Pathogenic Avian Influenza Virus Strains in Chicken and the Differences in Sensitivity among Different Chicken Breeds

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.