Enrichment of corneal epithelial stem/progenitor cells using cell surface markers, integrin alpha6 and CD71

Corneal epithelial stem cells are believed to reside in the basal layer of the limbal epithelium, but no definitive cell surface markers have been identified. For keratinocytes, stem/progenitor cells are known to be enriched by cell surface markers, integrin α₆ and CD71, as a minor subpopulation which shows high integrin α₆ and low CD71 expressions (α₆^bri/CD71^dim). In the present study, we investigated the possibility that corneal epithelial stem cells can be enriched by integrin α₆ and CD71. The α₆^bri/CD71^dim cells were separated by fluorescence-activated cell sorting, as a minor subpopulation of the limbal epithelial cells. They were enriched for relatively small cells, showing a higher clonogenic capacity and expression of stem cell markers, but a lower expression of differentiation markers, compared to other cell populations. The cells were localized immunohistochemically in the basal region of the limbal epithelium. These results indicate that the α₆^bri/CD71^dim subpopulation enriched corneal epithelial stem cells.     

6-Shogaol and 6-gingerol, the pungent of ginger, inhibit TNF-alpha mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes

In this study, we demonstrated that the two ginger-derived components have a potent and unique pharmacological function in 3T3-L1 adipocytes via different mechanisms. Both pretreatment of 6-shogaol (6S) and 6-gingerol (6G) significantly inhibited the tumor necrosis factor-α (TNF-α) mediated downregulation of the adiponectin expression in 3T3-L1 adipocytes. Our study demonstrate that (1) 6S functions as a PPARγ agonist with its inhibitory mechanism due to the PPARγ transactivation, and (2) 6G is not a PPARγ agonist, but it is an effective inhibitor of TNF-α induced c-Jun-NH₂-terminal kinase signaling activation and thus, its inhibitory mechanism is due to this inhibitory effect.     

Conjugated docosahexaenoic acid inhibits lipid accumulation in rats

Conjugated linoleic acid (CLA), which contains a conjugated double-bond system, and n-3 highly unsaturated fatty acids such as docosahexaenoic acid (DHA) are widely known to improve lipid metabolism. To examine the possibility that a fatty acid with a combination of these structural features might have stronger physiological effects, we prepared conjugated DHA (CDHA) by alkaline isomerization of DHA and examined its effects on lipid and sugar metabolism in rats. Rats were force fed with 200 mg of test oils [linoleic acid (LA), DHA, CLA or CDHA] everyday for 4 weeks. Compared with the animals from the other groups, those in the CDHA group showed a significant weight loss in white adipose tissue (57% of adipose tissue weight in the LA group) and significant decreases in the levels of liver triacylglycerol (TG; 65% of TG level in the LA group) as well as total cholesterol (TC; 88% of TC level in the LA group), indicating suppression of lipid accumulation in the liver and adipose tissue. In addition, plasma TG and TC levels significantly decreased (69% of TG level and 82% of TC level in the LA group), indicating improved lipid metabolism. In the liver, the fatty acid synthesis system was inhibited and the fatty acid beta-oxidation system was activated, whereas the free fatty acid, glucose and tumor necrosis factor alpha levels in the plasma were lowered following CDHA administration. Hence, intake of CDHA appears to suppress the accumulation of fat in the liver and epididymal adipose tissue and improves lipid and sugar metabolism in rats.     

The molecular phylogeny of the genus Rhizopus based on rDNA sequences

In order to establish the molecular phylogeny of the genus Rhizopus, three molecules of the ribosomal RNA-encoding DNA (rDNA), complete 18S, internal transcribed spacer (ITS)1-5.8S-ITS2, and 28S D1/D2 regions of all the species of the genus were sequenced. Phylogenetic trees showed three major clusters corresponding to the three groups in the current morphological taxonomy, microsporus-group, stolonifer-group, and R. oryzae. R. stolonifer var. lyococcos was clustered independently from the major clusters. R. schipperae clustered differently in all trees. Strains of R. sexualis had multiple ITS sequences. A. rouxii clustered with R. oryzae. These results indicate the possibility of molecular identification of species groups using rDNA sequencing. Reclassification of the genus might be appropriate.     

CD8+ T cells play disparate roles in the induction and the effector phases of murine experimental allergic conjunctivitis

Although CD4+ Th2 cells clearly play an essential role in the development of experimental allergic diseases, the functions CD8+ T cells may have in these diseases have been investigated less extensively and remain controversial. Here, we investigated the roles of CD8+ T cells in the development of experimental allergic conjunctivitis (EC). EC was induced in CD8alpha-deficient (CD8KO) mice and wild-type (WT) mice by active immunization with short ragweed pollen (RW) followed by challenge with RW-containing eye drops. Alternatively, EC was induced by transferring RW-primed splenocytes followed by RW challenge. With regard to actively immunized mice, CD8KO mice showed significantly less severe eosinophil infiltration of the conjunctiva and lower total IgE levels, although the levels of the other Igs were equivalent between the two strains. Cytokine production by cultured splenocytes also did not differ, but the WT conjunctivas showed upregulated IL-5 and IL-6 expression and greater upregulation of IL-4 expression than the conjunctivas of CD8KO mice. Thus, CD8+ T cells may play a significant role during the induction phase by aiding IgE production and the generation of Th2 cytokines in the conjunctiva, thus promoting the development of EC. In contrast, splenocytes from CD8KO mice induced significantly more severe EC in WT mice than cells from WT mice. In addition, transfer of RW-primed splenocytes induced significantly more severe eosinophil infiltration in CD8KO recipient mice. Thus, CD8+ T cells promote the development of EC during the induction phase, but suppress it during the effector phase.     

Nervostroma, gen. nov. in the Sclerotiniaceae, the teleomorph of Cristulariella, and Hinomyces anam. gen. nov. to accommodate the anamorph of Grovesinia: reassessment of the genus Cristulariella

Taxonomy of the genus Cristulariella is revised, retaining Cristulariella (Crist.) depraedans as the type. Two new species, Crist. cercidiphylli and Crist. corni, are additionally described under the genus. The new anamorphic genus Hinomyces is erected to accommodate Botrytis (Cristulariella) moricola and Cristulariella pruni. A new genus and species, Nervostroma depraedans, is erected in the Sclerotiniaceae to accommodate the teleomorph of Crist. depraedans, with an additional species, Nervostroma cercidiphylli.     

LC-MS/MS analysis of carboxymethylated and carboxyethylated phosphatidylethanolamines in human erythrocytes and blood plasma

An amino group of phosphatidylethanolamine (PE) is considered as a target for nonenzymatic glycation, and the potential involvement of lipid glycation in the pathogenesis of diabetic complications has generated interest. However, unlike an early glycation product of PE (Amadori-PE), the occurrence and roles of advanced glycation end products of PE (AGE-PE) in vivo have been unclear. Here, we developed an LC-MS/MS method for the analysis of AGE-PE [carboxymethyl-PE (CM-PE) and carboxyethyl-PE (CE-PE)]. Collision-induced dissociation of CM-PE and CE-PE produced characteristic ions, permitting neutral loss scanning (NLS) and multiple reaction monitoring (MRM) of AGE-PE. By NLS analysis, a series of AGE-PE molecular species was detected in human erythrocytes and blood plasma. In LC-MS/MS analysis, MRM enabled the separation and determination of the predominant AGE-PE species. Between healthy subjects and diabetic patients, no significant differences were observed in AGE-PE concentrations in erythrocytes and plasma, whereas Amadori-PE concentrations were higher in diabetic patients. These results provide direct evidence for the presence of AGE-PE in human blood, and indicated that, compared with Amadori-PE, AGE-PE is less likely to be accumulated in diabetic blood. The presently developed LC-MS/MS method appears to be a powerful tool for understanding in vivo lipid glycation and its pathophysiological consequence.     

Potential of selected strains of lactic acid bacteria to induce a Th1 immune profile

Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.     

Rhizoid differentiation of <Emphasis Type="Italic">Spirogyra</Emphasis> is regulated by substratum

Some species of Spirogyra can anchor to substratum with rod- or rosette-shaped rhizoid (hapteron). The rhizoid differentiation can be induced by cutting algal filaments in a laboratory. Requirement of contact stimulation for rhizoid differentiation has been reported (Nagata in Plant Cell Physiol 14:531-541, 1973a). However, the control mechanism of rhizoid morphology has not been elucidated. When cut filaments were incubated on the glass surface, start of tip growth, secretion of lectin-binding material and callose synthesis were observed. In the absence of contact to the glass surface, none of above phenomena was induced. Systematic analysis showed that rosette-shaped rhizoid was formed only on the hydrophobic substratum. On the hydrophobic substratum, both Bandeiraea (Griffonia) simplicifolia lectin and jacalin strongly stained the rhizoids. On the hydrophilic substratum, however, only Bandeiraea (Griffonia) simplicifolia lectin strongly stained the rhizoids.