Effect of sugar alcohols on gut function and body composition in normal and cecectomized rats.

The effects of two sugar alcohols on feed utilization, digesta retention, gut fermentation and serum lipid profiles were compared in normal and cecectomized rats to examine the possibility of the cecectomized rat as an experimental animal with relevance to humans. Semi-purified diets containing no sugar alcohol, 7% sorbitol or 7% lactitol were fed to normal and cecectomized rats for 16 days. The digestibility of the crude fat and the compositions of the carcass dry matter and crude fat were significantly decreased by feeding sugar alcohols in both groups, but the effects were relatively higher in the cecectomized rats than in the normal rats. Diarrhea, faster transit times and shorter retention times of digesta were noted in the cecectomized rats fed sugar alcohols, while the inverse results were observed in the normal rats fed similar diets. The concentration of cecal organic acids was increased in the normal rats, whereas the concentration of colonic organic acids was decreased in the cecectomized rats fed sugar alcohols, compared with their corresponding control groups. The concentration of serum total cholesterol was decreased in both the normal and cecectomized rats fed diets containing sugar alcohols. The tendencies for diarrhea, faster digesta transit and reduced body fat induced by the fermentable materials in the cecectomized rat have good relevance to the parallel effects of fermentable materials in humans, suggesting the possibility of using the cecectomized rat as a model to study some of the physiological effects of sugar alcohols in humans.     

DJ-1 Protects Pancreatic Beta Cells from Cytokine- and Streptozotocin-Mediated Cell Death

A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson’s disease gene PARK7 protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined in vitro after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and in vitro inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.     

Utility of NucleoCounter for the chondrocyte count in the collagenase digest of human native cartilage

In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R² = 0.9999) and reproducibility (%CV: 2.00–8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R² = 0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20–1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions.     

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes

Objective:  Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation.
Materials and methods:  We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA).
Results:  FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-β), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-β expression, suggesting a decrease in responsiveness of TGF-β to FGF18 stimulation in the dedifferentiated chondrocytes.
Conclusion:  FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.     

Bayesian association mapping of multiple quantitative trait loci and its application to the analysis of genetic variation among <Emphasis Type="Italic">Oryza sativa</Emphasis> L. germplasms

One way to use a crop germplasm collection directly to map QTLs without using line-crossing experiments is the whole genome association mapping. A major problem with association mapping is the presence of population structure, which can lead to both false positives and failure to detect genuine associations (i.e., false negatives). Particularly in highly selfing species such as Asian cultivated rice, high levels of population structure are expected and therefore the efficiency of association mapping remains almost unknown. Here, we propose an approach that combines a Bayesian method for mapping multiple QTLs with a regression method that directly incorporates estimates of population structure. That is, the effects due to both multiple QTLs and population structure were included in our statistical model. We evaluated the efficiency of our approach in simulated- and real-trait analyses of a rice germplasm collection. Simulation analyses based on real marker data showed that our model could suppress both false-positive and false-negative rates and the error of estimation of genetic effects over single QTL models, indicating that our model has statistically desirable attributes over single QTL models. As real traits, we analyzed the size and shape of milled rice grains and found significant markers that may be linked to QTLs reported previously. Association mapping should have good prospects in highly selfing species such as rice if proper methods are adopted. Our approach will be useful for the whole genome association mapping of various selfing crop species.     

Four new crystal structures of Tk-subtilisin in unautoprocessed, autoprocessed and mature forms: insight into structural changes during maturation

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-subtilisin) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of the propeptide. The crystal structures of the autoprocessed and mature forms of Tk-subtilisin were determined at 1.89 A and 1.70 A resolution, respectively. Comparison of these structures with that of unautoprocessed Pro-Tk-subtilisin indicates that the structure of Tk-subtilisin is not seriously changed during maturation. However, one unique Ca(2+)-binding site (Ca-7) is identified in these structures. In addition, the N-terminal region of the mature domain (Gly70-Pro82), which binds tightly to the main body in the unautoprocessed form, is disordered and mostly truncated in the autoprocessed and mature forms, respectively. Interestingly, this site is formed also in the unautoprocessed form when its crystals are soaked with 10 mM CaCl(2), as revealed by the 1.87 A structure. Along with the formation of this site, the N-terminal region (Leu75-Thr80) is disordered, with the scissile peptide bond contacting with the active site. These results indicate that the calcium ion binds weakly to the Ca-7 site in the unautoprocessed form, but is trapped upon autoprocessing. We propose that the Ca-7 site is required to promote the autoprocessing reaction by stabilizing the autoprocessed form, in which the new N terminus of the mature domain is structurally disordered. Furthermore, the crystal structure of the Tk-propeptide:S324A-subtilisin complex, which was formed by the addition of separately expressed proteins, was determined at 1.65 A resolution. This structure is virtually identical with that of the autoprocessed form, indicating that the interaction between the two domains is highly intensive and specific.     

Chromosomal study of lettuce and its allied species (Lactuca spp., Asteraceae) by means of karyotype analysis and fluorescence in situ hybridization

In this study, in addition to the karyotype analysis, the chromosomal distributions of 5 S and 18 S rDNAs, and the Arabidopsis-type (T3AG3) telomeric sequences were detected by means of fluorescence in situ hybridization (FISH) to promote the information of chromosomal organization and evolution in the cultivated lettuce and its wild relatives, L. sativa, L. serriola, L. saligna and L. virosa. The karyotype analysis revealed the dissimilarity between L. virosa and the remaining species. In all four Lactuca species studied, one 5 S rDNA and two 18 S rDNA loci were detected. The simultaneous FISH of 5 S and 18 S rDNAs revealed that both rDNA loci of L. sativa, L. serriola and L. saligna were identical, however, that of L. virosa was different from the other species. These analyses indicate the closer relationships between L. sativa/L. serriola and L. saligna rather than L. virosa. Arabidopsis-type telomeric sequences were detected at both ends of their chromatids of all chromosomes not in the other regions. This observation suggests the lack of telomere-mediated chromosomal rearrangements among the Lactuca chromosomes.