Synchronized reconstitution of muscle fibers,peripheral nerves and blood vessels by murine skeletal muscle-derived CD34<Superscript>−</Superscript>/45<Superscript>−</Superscript> cells

In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution capacity of CD34⁻/CD45⁻ (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2–8 × 10⁴); however, the number increased 10–20 fold (2–16 × 10⁵) after 6 days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type) resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20% recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve (Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule) and fluorescence in situ hybridization (FISH) analysis.     

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNA later at 4 degrees C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.     

Truncated KCNQ1 mutant, A178fs/105, forms hetero-multimer channel with wild-type causing a dominant-negative suppression due to trafficking defect

We identified a novel mutation Ala178fs/105 missing S3-S6 and C-terminus portions of KCNQ1 channel. Ala178fs/105-KCNQ1 expressed in COS-7 cells demonstrated no current expression. Co-expression with wild-type (WT) revealed a dominant-negative effect, which suggests the formation of hetero-multimer by mutant and WT. Confocal laser microscopy displayed intracellular retention of Ala178fs/105-KCNQ1 protein. Co-expression of the mutant and WT also increased intracellular retention of channel protein compared to WT alone. Our findings suggest a novel mechanism for LQT1 that the truncated S1-S2 KCNQ1 mutant forms hetero-multimer and cause a dominant-negative effect due to trafficking defect.     

Functional Analysis of Fructosyl-Amino Acid Oxidases of Aspergillus oryzae

Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N-fructosyl N^α-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain ΔfaoAo2 did not grow. Addition of glucose or (NH₄)₂SO₄ to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO₂ as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.     

Altered kinetics of pituitary response to gonadotropin-releasing hormone in women with variant luteinizing hormone: correlation with ovulatory disorders

OBJECTIVE: The LH response of pituitary gland to gonadotropin-releasing hormone (GnRH) stimulation is not well defined in patients with mutant beta-subunit (Trp(8) to Arg(8) and Ile(15) to Thr(15)). Here we compared the relative activities and dynamics of LH secretion in patients with wild-type and variant LH following injection of GnRH. METHODS: A GnRH stimulation test was performed in 33 patients with ovulatory disorders (patient group) and 29 women with normal ovulatory cycles (control group) heterozygous for the variant LHbeta allele. Blood samples were obtained up to 120 min after GnRH injection. Serum LH response was determined by comparing the results of LH immunoassays using a monoclonal antibody that recognizes wild-type LH only with those of another assay using a polyclonal antibody that recognizes equally both variant and wild-type LH (total LH). The ratio of variant LH to total LH (LH ratio) was used to determine the serum LH status. RESULTS: The LH ratio in the control group showed the peak 15 min after GnRH injection, while that in the patient group showed the peaks 30-60 min after injection. The LH ratio in the patient group at 120 min after injection was significantly lower than that in the control group. The percent increases in LH ratio in both groups showed the peak 15 min after injection. The patient group had significantly lower changes of LH ratio at 15, 60, 90 and 120 min after GnRH injection compared with that in the control group. CONCLUSION: Differences in circulatory kinetics of the two types of LH may explain the differences in LH function between patients with ovulatory disorders and women with normal ovulatory cycles.     

Sex change of primary males in a diandric labrid Halichoeres trimaculatus: coexistence of protandry and protogyny within a species

In the threespot wrasse Halichoeres trimaculatus , sex change of primary males was observed in the field and confirmed by aquarium experiments. In other words, protandry and protogyny coexisted within this species. Moreover, male-to-female-to-male sex change and female-to-male-to-female sex change were observed in aquarium experiments; i.e . reversed sex change occurred in both protandrous and protogynous hermaphrodites. These results suggest that only the direction of sex differentiation before maturation may differ between the two sexual types that have been regarded as a primary male and a protogynous hermaphrodite.     

Anti‐biofilm and bactericidal effects of magnolia bark‐derived magnolol and honokiol on Streptococcus mutans

Dental caries affects people of all ages and is a worldwide health concern. Streptococcus mutans is a major cariogenic bacterium because of its ability to form biofilm and induce an acidic environment. In this study, the antibacterial activities of magnolol and honokiol, the main constituents of the bark of magnolia plants, toward planktonic cell and biofilm of S. mutans were examined and compared with those of chlorhexidine. The minimal inhibitory concentrations of magnolol, honokiol and chlorhexidine for S. mutans were 10, 10 and 0.25 µg/mL, respectively. In addition, each agent showed bactericidal activity against S. mutans planktonic cells and inhibited biofilm formation in a dose‐ and time‐dependent manner. Magnolol (50 µg/mL) had greater bactericidal activity against S. mutans biofilm than honokiol (50 µg/mL) and chlorhexidine (500 µg/mL) at 5 min after exposure, while all showed scant activity against biofilm at 30 s. Furthermore; chlorhexidine (0.5–500 µg/mL) exhibited high cellular toxicity for the gingival epithelial cell line Ca9‐22 at 1 hr, whereas magnolol (50 µg/mL) and honokiol (50 µg/mL) did not. Thus; it was found that magnolol has antimicrobial activities against planktonic and biofilm cells of S. mutans. Magnolol may be a candidate for prevention and management of dental caries.     

Chlorination of harman and norharman with sodium hypochlorite and co-mutagenicity of the chlorinated products

Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water. It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o-toluidine with S9 mix. When these β-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced. Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o-toluidine toward Salmonella typhimurium TA98 with S9 mix. These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.     

Four mutually incompatible Argentine ant supercolonies in Japan: inferring invasion history of introduced Argentine ants from their social structure

In recent years, highly invasive ant species successively invaded warm regions of Asia. In Japan, the Argentine ant, Linepithema humile, has become established in several coastal regions. This species forms unusual social organizations called supercolonies consisting of numerous mutually non-aggressive nests. We studied the behavioral relationships, similarity of cuticular hydrocarbon profiles (nestmate recognition cue), and genetic relationships among the introduced Argentine ant populations of Japan. The Japanese populations were divided into four behaviorally, chemically, and genetically distinct supercolonies, which may have derived from independent source populations. The result represents the recent trend of increasing invasions of invasive ants to Asia. The discontinuous distribution of one supercolony throughout most of the Japanese range suggests rapid expansion of the supercolony via human-mediated jump dispersal. Meanwhile, localization of the other three supercolonies in Kobe Port provides the first evidence for multiple invasions of distinct supercolonies into a base for international trade.