Purification and characterization of two phospho-β-galactosidases, LacG1 and LacG2, from Lactobacillus gasseri ATCC33323(T)

Lactobacillus gasseri ATCC33323(T) expresses four enzymes showing phospho-β-galactosidase activity (LacG1, LacG2, Pbg1 and Pbg2). We previously reported the purification and characterization of two phospho-β-galactosidases (Pbg1 and Pbg2) from Lactobacillus gasseri JCM1031 cultured in lactose medium. Here we aimed to characterize LacG1 and LacG2, and classify the four enzymes into 'phospho-β-galactosidase' or 'phospho-β-glucosidase.' LacG1 and recombinant LacG2 (rLacG2), from Lb. gasseri ATCC33323(T), were purified to homogeneity using column chromatography. Kinetic experiments were performed using sugar substrates, o-nitrophenyl-β-D-galactopyranoside 6-phosphate (ONPGal-6P) and o-nitrophenyl-β-D-glucopyranoside 6-phosphate (ONPGlc-6P), synthesized in our laboratory. LacG1 and rLacG2 exhibited high k(cat)/K(m) values for ONPGal-6P as compared with Pbg1 and Pbg2. The V(max) values for ONPGal-6P were higher than phospho-β-galactosidases previously purified and characterized from several lactic acid bacteria. A phylogenetic tree analysis showed that LacG1 and LacG2 belong to the phospho-β-galactosidase cluster and Pbg1 and Pbg2 belong to the phospho-β-glucosidase cluster. Our data suggest two phospho-β-galactosidase, LacG1 and LacG2, are the primary enzymes for lactose utilization in Lb. gasseri ATCC33323(T). We propose a reclassification of Pbg1 and Pbg2 as phospho-β-glucosidase.     

Phase-dependent locomotor activity in first-stadium nymphs of the desert locust, Schistocerca gregaria: effects of parental and progeny rearing density

This study examined the effects of parental and progeny rearing density on locomotor activity of 1st-stadium nymphs of the desert locust, Schistocerca gregaria, using an actograph. Progeny obtained from solitarious (isolated-reared) or gregarious (crowd-reared) locusts were reared in isolation or in a group of 30 nymphs. Crowding after hatching had a slight influence on mean activity shortly after the start of measurements, but no clear effect was detected until day 2, when maximum activity during the 6-24 h of observation was significantly higher than that of the nymphs kept in isolation. On the other hand, the effects of parental rearing density on locomotor activity manifested at all ages examined (0-2 days old). Progeny of gregarious locusts showed consistently higher activity than those of solitarious locusts. In newly hatched nymphs, the effect of parental rearing density was explained by variation in body size at hatching, one of the phase-dependent characteristics. Hatchling body color was also correlated with locomotor activity and body weight. Similar levels of locomotor activity were exhibited when green, solitarious and black, gregarious nymphs were similar in body weight. These results suggested that parental rearing density indirectly influences locomotor activity in the progeny shortly after hatching by affecting their body size as eggs or hatchlings.     

Characterization of Paecilomycescinnamomeus from the camellia whitefly, Aleurocanthus camelliae (Hemiptera: Aleyrodidae), infesting tea in Japan

The whitefly, Aleurocanthus camelliae Kanmiya and Kasai (Hemiptera: Aleyrodidae), is an invasive species in Japan that was first discovered in 2004 on tea in Kyoto. Soon after its arrival epizootics of an entomopathogenic fungus were observed in populations of the whitefly in many tea-growing regions. Here we identify this fungus as Paecilomyces cinnamomeus (Petch) Samson and W. Gams (Hypocreales: Clavicipitaceae) based on morphological characteristics and molecular analyses. This is the first record of P. cinnamomeus in Japan and also the first time it has been recorded from the genus Aleurocanthus. A isolate of P. cinnamomeus caused greater than 50% and 90% infection in whitefly nymphs at 1 × 10⁶ and 1 × 10⁷ conidia/ml respectively, while the commercial mycoinsecticides Preferd® (Isaria fumosorosea) and Mycotal® (Lecanicillium muscarium) caused <10% infection at their recommended field rates (5 × 10⁶ and 9 × 10⁶ conidia/ml, respectively), suggesting that P. cinnamomeus may be more useful as a control agent than the currently available mycoinsecticides. Optimum and upper limit temperatures for in vitro growth of P. cinnamomeus isolates were 22.5–25 °C and 32.5 °C, respectively. At field rates, the fungicide thiophanate-methyl caused some inhibition of in vitro growth of P. cinnamomeus isolates, and the bactericide copper oxychloride and the insecticides tolfenpyrad and methidathion were strongly inhibitory. The findings obtained in this study will be useful in the development of microbial control programs using P. cinnamomeus against A. camelliae.     

Antibodies to pathogenic epitopes on type XVII collagen cause skin fragility in a complement-dependent and -independent manner

In bullous pemphigoid (BP), the most prevalent autoimmune blistering disease, type XVII collagen (COL17) is targeted by circulating autoantibodies. BP is thought to be an autoantibody-mediated complement-fixing blistering disease, and a juxtamembranous noncollagenous 16A (NC16A) domain spanning Glu(490) to Arg(566) was proved to be the main pathogenic region on COL17, although precise pathogenic epitopes within NC16A have not been elucidated. In this study, we showed that injection of rabbit IgG Abs targeting Asp(522) to Gln(545) induced skin fragility associated with in vivo deposition of IgG and complement in neonatal COL17-humanized mice. Notably, immunoadsorption of rabbit anti-NC16A IgG Ab with this epitope (Asp(522) to Gln(545)) or the anti-NC16A IgG administered together with the peptides of this epitope as a decoy ameliorated skin fragility in the injected neonatal COL17-humanized mice compared with the anti-NC16A IgG alone even though all of the mice showed both IgG and complement deposition. These results led us to investigate an additional, complement-independent mechanism of skin fragility in the mice injected with anti-COL17 Abs. The rabbit anti-NC16A IgG depleted the expression of COL17 in cultured normal human keratinocytes, whereas immunoadsorption of the same IgG with this epitope significantly suppressed the depletion effect. Moreover, passive transfer of F(ab')(2) fragments of the human BP or rabbit IgG Abs against COL17 demonstrated skin fragility in neonatal COL17-humanized mice. In summary, this study reveals the importance of Abs directed against distinct epitopes on COL17, which induce skin fragility in complement-dependent as well as complement-independent ways.     

Depletion of invariant NKT cells reduces inflammation-induced preterm delivery in mice

This study sought to determine whether invariant NKT (iNKT) cells play an essential role in inflammation-induced preterm delivery. Preterm delivery and fetal death rates were determined in wild-type (WT) C57BL/6 mice and iNKT cell-deficient Jα18(-/-) mice injected i.p. with LPS. The percentages of decidual immune cells, including activated subsets, and costimulatory molecule expression were analyzed by flow cytometry. Th1 and Th2 cytokine production in the culture supernatants of decidual mononuclear cells was measured by ELISA. To some extent, Jα18(-/-) mice were resistant to LPS-induced preterm delivery. The proportions of decidual CD3(+) and CD49b(+) cells were slightly lower in Jα18(-/-) mice than in WT Jα18(+/+) mice, whereas almost no CD3(+)CD49b(+) cells could be found in Jα18-null mice. The percentages of activated decidual DCs, T cells, and NK cells were significantly lower in LPS-treated Jα18(-/-) mice than in WT mice. The CD40, CD80, and CD86 expression levels on decidual CD11c(+) cells from Jα18(-/-) mice were also significantly lower than in WT mice. Mean concentrations of Th1 cytokines IFN-γ and IL-12p70 in the culture supernatants of decidual mononuclear cells from LPS-treated Jα18(-/-) mice were apparently lower than those of LPS-induced WT mice. Additionally, the proportions of activated CD11c(+) cells, CD3(+) cells, and CD49b(+) cells in LPS-induced preterm delivery mice were strikingly higher in both WT and null mice when compared with the control PBS group and LPS-injected but normally delivered mice. Our results suggest that iNKT cells may play an essential role in inflammation-induced preterm birth.     

Elevation of GM2 ganglioside during ethanol-induced apoptotic neurodegeneration in the developing mouse brain

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase 3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase 3 activation in the 7-day-old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration.     

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.     

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.     

Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.     

The planarian P2X homolog in the regulation of asexual reproduction

The growth in size of freshwater planarians in response to nutrient intake is limited by the eventual separation of tail and body fragments in a process called fission. The resulting tail fragment regenerates the entire body as an artificially amputated tail fragment would do, and the body fragment regenerates a tail, resulting in two whole planarians. This regenerative ability is supported by pluripotent somatic stem cells, called neoblasts, which are distributed throughout almost the entire body of the planarian. Neoblasts are the only planarian cells with the ability to continuously proliferate and give rise to all types of cells during regeneration, asexual reproduction, homeostasis, and growth. In order to investigate the molecular characteristics of neoblasts, we conducted an extensive search for neoblast-specific genes using the High Coverage Expression Profiling (HiCEP) method, and tested the function of the resulting candidates by RNAi. Disruption of the expression of one candidate gene, DjP2X-A (Dugesia japonica membrane protein P2X homologue), resulted in a unique phenotype. DjP2X-A RNAi leads to an increase of fission events upon feeding. We confirmed by immunohistochemistry that DjP2X-A is a membrane protein, and elucidated its role in regulating neoblast proliferation, thereby explaining its unique phenotype. We found that DjP2X-A decreases the burst of neoblast proliferation that normally occurs after feeding. We also found that DjP2X-A is required for normal proliferation in starved animals. We propose that DjP2X-A modulates stem cell proliferation in response to the nutritional condition.