Alteration of apoptotic protease-activating factor-1 (APAF-1)-dependent apoptotic pathway during development of rat brain and liver.

Brain and liver extracts of rats at different stages after birth were examined for cytochrome c/dATP-dependent caspase (DEVDase)-activation (mitochondria pathway) in vitro. The caspase-activating activity in the brain extracts rapidly decreased after birth, reaching approximately 50 and 5%, at 1 and 2 weeks, respectively, of that in a 3-days- newborn sample, and essentially no caspase-activation was detected in the adult rat brain extracts. Such a dramatic change was not detected in the liver samples, suggesting that the observed abrogation of the cytochrome c-dependent mitochondria pathway after birth is a brain-specific event. In order to determine the factor(s) lacking in adult brain, we separately measured Apaf-1, procaspase 9, and pro-DEVDase activities using a supplementation assay. In adult brain, Apaf-1 activity was scarcely detected, while the tissue retained low but significant amounts of procaspase 9 (16% of that in the fetal tissue) and a pro-DEVDase (3.4%). In contrast, adult liver extracts retained relatively high levels of all of these factors. Immunoblot analyses clearly indicated that the expression of Apaf-1 and procaspase 3 is markedly suppressed within 4 weeks after birth in brain tissue while they are even expressed in adult liver. Considering these results together, we propose that, in the brain, the cytochrome c-dependent mitochondria pathway, which is essential for the programmed cell death during normal morphogenesis, is abrogated within 2-4 weeks after birth, whereas the pathway is still active in other adult tissues such as liver.     

Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli.

We previously reported that overexpression of the soxS or robA gene causes in several Escherichia coli strains the acquisition of higher organic solvent tolerance and also increased resistance to a number of antibiotics (H. Nakajima, K. Kobayashi, M. Kobayashi, H. Asako, and R. Aono, Appl. Environ. Microbiol. 61:2302-2307, 1995). Most E. coli strains cannot grow in the presence of cyclohexane. We isolated the marRAB genes from a Kohara lambda phage clone and cyclohexane-tolerant mutant strain OST3408. We found a substitution of serine for arginine at position 73 in the coding region of marR of OST3408 and designated the gene marR08. Our genetic analysis revealed that marR08 is responsible for the cyclohexane-tolerant phenotype. We observed that the marA gene on high-copy-number plasmids increased the organic solvent tolerance of E. coli strains. Furthermore, exposure of E. coli cells to salicylate, which activates the mar regulon genes, also raised organic solvent tolerance. Overexpression of the marA, soxS, or robA gene increased resistance to numerous antibiotics but not to hydrophilic aminoglycosides.     

in Vitro Effect of Deoxynivalenol on the Differentiation of Human Colonic Cell Lines Caco-2 and t84

The effects of low concentrations of deoxynivalenol (DON) on structural and functional characteristics of human colonic adenocarcinoma cell lines Caco-2 and T84 were examined. Scanning electron microscopic (SEM) analysis of the apical surfaces of Caco-2 cells revealed reduction or abnormal formation of brush borders in the presence of 50, 100 and 200 ng/ml of DON. Monolayer integrity of Caco-2 and T84 cells was studied using cells which were cultured on permeable membranes. The transepithelial electrical resistance (TEER) of Caco-2 cells was significantly reduced at 50, 100 and 200 ng/ml of DON, significant increase in lucifer yellow (LY) permeability was also observed in these cells at 100 ng/ml of DON. The TEER of T84 cells was significantly reduced at 100 and 200 ng/ml of DON. LY permeability significantly increased at 200 ng/ml of DON in T84 cells. Enzyme activities in Caco-2 cells were also examined. Alkaline phosphatase activity was reduced from the 6th to 15th day of culture in the presense of 100 or 200 ng/ml of DON, whereas sucrase- isomaltase activity was significantly decreased by adding 50 or 100 ng/ml of DON for 15 or 20 days. Protein content was attenuated only by treatment with 200 ng/ml of DON thoughout the experimental period. The results indicate that DON interferes with structural and functional characteristics of differentiation in enterocytes at low doses. This revised version was published online in June 2006 with corrections to the Cover Date.     

The heterogeneity of human fibroblasts as determined from the effects of hydrocortisone on cell growth and specific dexamethasone binding

To elucidate the heterogeneity of human fibroblasts from lung and skin, the effects of hydrocortisone on cell proliferation and the specific dexamethasone binding to cells were studied. Hydrocortisone at physiological concentrations stimulated the proliferation in three strains of human fetal lung fibroblasts and inhibited it in two strains. There are two kinds of fibroblasts in the human fetal lung in addition to the human skin fibroblasts reported previously. Dexamethasone-binding experiments showed that human fibroblasts may be classified into two groups with respect to the dissociation constant (Kd) of the binding reaction. The heterogeneity of human fibroblasts shown by Kd could not be correlated to classification on the basis of the effects of hydrocortisone on cell proliferation. The differences in Kd for the binding reactions suggest differences in donor tissues from which human fibroblasts are derived.     

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.     

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-l-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.     

Insulin induces chloroquine-sensitive recycling of insulin-like growth factor II receptors but not of glucose transporters in rat adipocytes

Incubation of insulin-treated rat adipocytes with chloroquine, in a time- and concentration-dependent manner, was observed to inhibit the insulin-stimulated increase in insulin-like growth factor II (IGF-II) binding activity, whereas no significant change in IGF-II binding was observed in the absence of insulin. The incremental increase of insulin-stimulated IGF-II binding was inhibited 50% by 0.2 mM chloroquine within 15 min and was nearly completely abolished by 60 min. Interestingly, IGF-II binding was never observed to decrease below the binding value in cells without insulin treatment even when incubation was extended to 180 min. Scatchard analysis of IGF-II binding as well as the specific binding of an anti-IGF-II receptor antibody demonstrated that the loss of IGF-II binding in the insulin-stimulated chloroquine-treated adipocytes was due to a decrease in the number of cell-surface IGF-II receptors, whereas the total number of cellular IGF-II receptors was unaltered. The effect of chloroquine was observed to be reversible, temperature-dependent, and sensitive to the metabolic poison KCN. Furthermore, NH4Cl was also observed to inhibit insulin-stimulated increase in IGF-II binding. In contrast, chloroquine or NH4Cl did not inhibit the basal or insulin-stimulated glucose transport activity. Photoaffinity labeling of the glucose transporter with [3H]cytochalasin B also demonstrated that the basal and insulin-stimulated subcellular distribution of the glucose transporters was unaltered by chloroquine treatment. These results suggest that 1) insulin induces a constitutive, acidotropic agent-sensitive recycling of IGF-II receptor and 2) the glucose transporter and IGF-II receptor do not share the same insulin-regulated intracellular trafficking pathways.     

Flesh‐eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor‐κB ligand

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock‐like syndrome. Human CD46‐expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast‐mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 10⁵ colony‐forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor‐κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro‐computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4⁺ and CD8⁺ T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte‐derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast‐like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.     

Effect of Cetrorelix administration on ovarian stimulation in aged mice

In mice, ovarian stimulation via hormone administration is an effective method for obtaining many ova simultaneously, but its effect is reduced by the influence of aging. In this study, we demonstrate that this problem can be improved by administering the gonadotropin-releasing hormone antagonist Cetrorelix prior to ovarian stimulation. Before 12-month-old female mice were injected with 5 IU pregnant mare serum gonadotropin and 5 IU human chorionic gonadotropin, we administered 5 µg/kg Cetrorelix for 7 consecutive days (7 times) or 3 times once every 3 days. As a result, 8.7 ± 1.9 (mean ± SEM, n=10) and 9.8 ± 1.3 (n=10) oocytes were obtained, respectively, as opposed to 4.7 ± 1.2 oocytes (n=9) in the case of no administration. Collagen staining of ovarian tissue showed that Cetrorelix administration reduced the degree of fibrosis, which improved ovarian function. In addition, equivalent fertilization and fetal development rates between control and Cetrorelix-treated aged mouse-derived oocytes were confirmed by in vitro fertilization and embryo transfer (Fertilization rate; control: 92.2% vs. 3 times: 96.9%/7 times: 88.5%, Birth rate; control: 56.4% vs. 3 times: 58.3%/7 times: 51.8%), indicating the normality of the obtained oocytes. It is concluded that Cetrorelix improved the effect of superovulation in aged mice without reducing oocyte quality. This procedure will contribute to animal welfare by extending the effective utilization of aged female breeding mice.