Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.     

Protective effect of antiganglioside antibodies against experimental Trypanosoma brucei infection in mice

Liposome-associated ganglioside antigens (ganglioside GM1 or bovine brain gangliosides) were prepared to facilitate the potential protective efficacy for Trypanosoma brucei. Mice were immunized with liposome-associated ganglioside GM1 or bovine brain gangliosides intraperitoneally (i.p.). After immunization, significantly higher antigen-specific IgG and IgM antibodies were detected in sera than in the nonimmunized control group. When sera from immunized mice were analyzed for isotype distribution, antigen-specific IgG1, IgG2a, and IgG3 antibody responses were also noted. After immunization, mice were challenged i.p. with 1 x 10(2) cells of T. brucei. Sixty percentage of liposome-associated ganglioside GM1-immunized mice survived the infection, and all the mice immunized with bovine brain gangliosides-containing liposomes survived. However, all control mice died within 7 days after infection. These data demonstrate that liposomes containing ganglioside antigens have the potential usefulness for the induction of a protective immune response against T. brucei infection and suggest the possibility of developing vaccines that may ultimately be used for the prevention of trypanosomiasis.     

Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M1 and M3 muscarinic receptors

Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R-/-) or M3 (M3R-/-) receptor or that lacked both receptors (M(1/3)R-/-) to determine the role of M1R and M3R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R-/- mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R-/- with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M(1/3)R-/- double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M(1/3)R-/- mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.     

17-AAG, an Hsp90 inhibitor, ameliorates polyglutamine-mediated motor neuron degeneration

Heat-shock protein 90 (Hsp90) functions as part of a multichaperone complex that folds, activates and assembles its client proteins. Androgen receptor (AR), a pathogenic gene product in spinal and bulbar muscular atrophy (SBMA), is one of the Hsp90 client proteins. We examined the therapeutic effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), a potent Hsp90 inhibitor, and its ability to degrade polyglutamine-expanded mutant AR. Administration of 17-AAG markedly ameliorated motor impairments in the SBMA transgenic mouse model without detectable toxicity, by reducing amounts of monomeric and aggregated mutant AR. The mutant AR showed a higher affinity for Hsp90-p23 and preferentially formed an Hsp90 chaperone complex as compared to wild-type AR; mutant AR was preferentially degraded in the presence of 17-AAG in both cells and transgenic mice as compared to wild-type AR. 17-AAG also mildly induced Hsp70 and Hsp40. 17-AAG would thus provide a new therapeutic approach to SBMA and probably to other related neurodegenerative diseases.     

Polymorphism of cellulose I family: reinvestigation of cellulose IVI

Polymorphs of cellulose I, III(I), and IV(I) have been investigated by X-ray diffraction, FT-IR, and solid-state (13)C NMR spectroscopy. Highly crystalline cellulose III(I) samples were prepared by treating cellulose samples in supercritical ammonia at 140 degrees C for 1 h, and conventional cellulose III(I) samples were prepared by liquid ammonia treatment. The cellulose IV(I) sample of highest crystallinity was that prepared from Cladophora cellulose III(I) in supercritical ammonia, followed by the sample treated in glycerol at 260 degrees C for 0.5 h, whereas the lowest crystallinity was observed in ramie cellulose prepared by conventional liquid ammonia treatment followed by glycerol annealing. In general, the perfection of cellulose IV(I) depends on the crystallinity of the original material: either of the starting cellulose I or of the cellulose III(I) after ammonia treatment. The product thus obtained was analogous to cellulose I(beta), which is what it should be called rather than cellulose IV(I). If the existence of the polymorph cellulose IV(I) is not accepted, the observations on which it has been based may be explained by the fact that the structure termed cellulose IV(I) is cellulose I(beta) which contains lateral disorder.     

Expression of three mitochondrial solute carriers, citrin, aralar1 and ornithine transporter, in relation to urea cycle in mice

The present report describes the expression profiles of different tissues and developmental changes of mouse aspartate/glutamate carrier (AGC) genes, Slc25a13 and Slc25a12, and an ornithine transporter gene, Ornt1, in relation to urea cycle enzyme genes, carbamoylphosphate synthetase I (CPS) and argininosuccinate synthetase (ASS). Slc25a13 encodes citrin, recently found to be deficient in adult-onset type II citrullinemia and to function as AGC together with its isoform and product of Slc25a12, aralar1. Citrin was broadly distributed, but mainly in the liver, kidney and heart. Aralar1 was expressed in diaphragm, skeletal muscle, heart, brain and kidney, but not in the liver. These distribution profiles are different from the restricted of Ornt1, ASS and CPS. Citrin, ASS, CPS and Ornt1 showed similar patterns of developmental changes in the liver and small intestine, where they play a role in urea and arginine synthesis. Dietary, hormonal and physical manipulations caused varied changes of CPS, ASS and Ornt1 in the liver, but the change of citrin was not so marked as that of the others. Analysis using RT-PCR and restriction enzyme digestion revealed that the ornithine transporter most expressed is Ornt1, although Ornt2 is detectable at a minute level. All these results suggest that citrin as AGC plays a role in urea synthesis as well as many fundamental metabolic pathways in the liver, and shares metabolic functions with aralar1 in other tissues, and that Ornt1 is an important component in urea synthesis in the liver and in arginine synthesis in the small intestine during the neonatal period.     

Detection of anthrax spores from the air by real-time PCR

AIMS: To detect and isolate Bacillus anthracis from the air by a simple and rapid procedure. METHODS AND RESULTS: One hundred litres of air were filtered through an air monitor device. After the membrane was suspended in PBS, spores of B. anthracis were added. The suspension was plated on Bacillus cereus selective agar (BCA) plates to detect B. anthracis colonies. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using a Light Cycler system and anthrax-specific primers. CONCLUSION: A single cell of B. anthracis was detected by real-time PCR within 1 h and was also isolated on a BCA plate within two d. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR and a Light Cycler provides a flexible and powerful tool to prevent epidemics.