Functional analysis of Bifidobacterium genes is essential for understanding host-Bifidobacterium interactions with beneficial effects on human health; however, lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the plasmid replication gene repA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was introduced into this integrant in order to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by the plasmid incompatibility. The proposed system was confirmed to work as expected in B. longum 105-A using the chromosomal full-length β-galactosidase gene as a target. Then, markerless gene deletion was tested using the aga gene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and four out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of aga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-complemented strain. These functional analyses revealed that aga is the only gene encoding a functional α-galactosidase enzyme in B. longum 105-A.

Ginseng roots (Panax ginseng CA Meyer) have been used traditionally for the treatment, especially prevention, of various diseases in China, Korea, and Japan. Both experimental and clinical studies suggest ginseng roots to have pharmacological effects in patients with life-style-related diseases such as non-insulin-dependent diabetic mellitus, atherosclerosis, hyperlipidemia, and hypertension. The topical use of ginseng roots to treat skin complaints including atopic suppurative dermatitis, wounds, and inflammation is also described in ancient Chinese texts; however, there have been relatively few studies in this area. In the present paper, we describe introduce the biological and pharmacological effects of ginsenoside Rb(1) isolated from Red ginseng roots on skin damage caused by burn-wounds using male Balb/c mice (in vivo) and by ultraviolet B irradiation using male C57BL/6J and albino hairless (HR-1) mice (in vivo). Furthermore, to clarify the mechanisms behind these pharmacological actions, human primary keratinocytes and the human keratinocyte cell line HaCaT were used in experiments in vitro.

Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cutaneous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner.

HASH(0xf766a20)

Although signal transducer and activator of transcription 3 (Stat3) plays crucial roles in the determination of neural stem cell (NSC) fate, Stat3 has multiple roles in NSC function. Moreover, Stat3 plays important roles in neuronal survival and tumorigenesis. To investigate the overall effects of Stat3 on NSC fate, NSC were isolated from Stat3(flox/flox) mouse embryos (E14-15d), in which both Stat3 alleles are flanked by LoxP sites. Isolated NSC were inoculated with an adenovirus vector expressing Cre recombinase (Ad.nCre) or a control adenovirus vector expressing beta-galactosidase (Ad.nLz). Three days later, quantitative real-time PCR (qPCR) analysis revealed that treatment with Ad.nCre eliminated stat3 mRNA expression in NSC. Promoter assay confirmed that overexpression of nCre inhibited transactivation of acute responsive element (APRE) and blocked Stat3 function in NSC. Moreover, western blot analysis and immunocytochemical analysis revealed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis. In addition, we investigated the effects of Stat3 elimination in NSC on the mRNA expression of Notch family members and bHLH factors. Consequently, qPCR analysis showed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis through down-regulation of notch1, notch2 and hes5, but not hes1 mRNA expression.

We investigated IgE-mediated allergic responses in a metabolic syndrome model rat strain, SHRSP.Z, which develops obesity and hypertension to cast light on the relationship between metabolic disturbances and allergic responses. IgE-mediated cutaneous anaphylactic responses were severely attenuated in this strain regardless of the presence of fa/fa mutation, compared with the parental WKY/Izm strain. Furthermore, in the peritoneal mast cells of both the SHRSP.Z and SHRSP/Izm strains, IgE-mediated activation, such as degranulation and protein tyrosine phosphorylation, was severely impaired whereas no significant differences were found in morphology and number of peritoneal mast cells. Immunoblot analyses revealed that phosphorylation levels of Syk upon IgE-mediated antigen stimulation was significantly decreased and basal expression of linker for activation of T cells (LAT) was down-regulated in peritoneal mast cells of the SHRSP strains. These results suggest that attenuated cutaneous allergic responses in the SHRSP.Z strain might be attributed to impaired FcvarepsilonRI-mediated signal transduction in mast cells.

We investigated the effects of a high-cholesterol (HC) diet administered long term (25 or 55 weeks) on metabolic disorders including hepatic damage in mice. The mice were fed the HC diet (15 % milk fat, 1.5 % cholesterol and 0.1 % cholic acid, w/w) for 25 or 55 weeks. Body and adipose tissue weights were similar to those of mice fed a control diet. Consumption of the HC diet long term resulted in hypercholesterolaemia, hepatic steatosis and gallstones. In addition, focal nodular hyperplasia (FNH) and mild fibrosis of the liver developed in all mice fed the HC diet for 55 weeks. Plasma levels of monocyte chemoattractant protein (MCP)-1 were elevated, and the level of hepatic platelet-derived growth factor (PDGF)-B protein was increased in mice fed the HC diet compared with those fed the control diet. Thus, it seems likely that the liver fibrosis and FNH caused by the long-term consumption of a HC diet may be partly due to an elevation of plasma MCP-1 and hepatic PDGF expression.

OBJECTIVE: Recent studies have demonstrated that a variety of chemokine receptors are expressed in mast cells. We investigated the changes in mRNA expression of CXCRs in murine IL-3-dependent bone marrow-derived mast cells (BMMCs) to clarify how the CXCR expression is regulated in mast cells. METHODS: Expression of CXCR mRNA was measured by RNase protection assay. Functional expression of CXCRs was confirmed by monitoring intracellular Ca(2+) mobilization. RESULTS: CXCR4 mRNA expression was transiently induced in BMMCs in serum-dependent fashion and was completely suppressed upon IgE-mediated antigen stimulation. In contrast, CXCR5 mRNA expression was induced upon IgE-mediated antigen stimulation. Changes in the intracellular Ca(2+) mobilization induced by CXCL12 strongly indicated the functional expression of CXCR4. The decrease in CXCR4 and the increase in CXCR5 mRNA expression was also observed in BMMCs stimulated with thapsigargin, a phorbol ester, and stem cell factor. CONCLUSION: The mRNA expression of CXCR4 is differentially regulated in BMMCs upon various stimuli including IgE-mediated antigen stimulation.

Oral ulcerative mucositis induced by chemotherapy or radiotherapy has an impact on quality of life, is dose-limiting for chemotherapy, and causes considerable morbidity. The aim of this study was to evaluate the effect of ginsenoside Rb1 on 5-fluorouracil (5-FU)-induced experimental oral mucositis in hamsters. Oral mucositis was induced in hamsters through a combination of 5-FU treatment and mild abrasion of the cheek pouch. Ginsenoside Rb1 isolated from ginseng was contained in chitosan-sodium alginate film (G-Rb1 film). The films were attached to the oral mucosa, and then the healing process was examined by measuring the area of mucositis, myeloperoxidase (MPO) activity and microscopic aspects. Films without ginsenoside Rb1 had no effect on 5-FU-induced oral mucositis in comparison to the control group. However, G-Rb1 films (10(-12)-10(-4) g/g film) dose-dependently improved recovery from 5-FU-induced damage, and there were significant differences between doses of 10(-6) and 10(-4) g/g film. These results suggest that topical application of films that contain ginsenoside Rb1 has a healing effect on severe oral mucositis induced by chemotherapy.

Syntaxin1 and synaptotagmin are located in the pre-synaptic terminals and play central roles in Ca(2+)-triggered neurotransmitter release. Because excessive synaptic transmission has been implicated in neuronal cell death after ischemia, we investigated the effects of cerebral ischemia on the levels of these proteins using a rat permanent focal ischemia model. Western blot analysis revealed that the protein level of syntaxin1 was significantly up-regulated in the ischemic core cortex and peri-ischemic cortex at 1 day after ischemia, while the protein level of synaptotagmin was not. Immunohistochemical analysis revealed that the protein level of syntaxin1 was markedly up-regulated in the ischemic areas where immunoreaction for MAP2 was lost. Furthermore, we showed that resident microglial cells were quite vulnerable to ischemia. Our data provide novel insights into the molecular mechanism of cerebral ischemia at the pre-synaptic terminals.