Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.     

Sparse and wavy hair: a new model for hypoplasia of hair follicle and mammary glands on rat chromosome 17

Mutant animals in the skin and hair have been used to identify important genes in biomedical research. We describe a new mutant rat, sparse and wavy hair (swh), that spontaneously arose in a colony of inbred WTC rats. The mutant phenotype was characterized by sparse and wavy hair, which was most prominent at age 3-4 weeks, and was inherited in an autosomal recessive manner. The swh/swh rats showed impaired gain of body weight, and their hair follicles were reduced both in number and size, associated with hypoplasia of the sebaceous glands and the subcutaneous fat tissue. Female swh/swh rats were unable to suckle their offspring. Their mammary glands were hypoplastic, and differentiation of mammary epithelial and myoepithelial cells was impaired. Linkage analysis of 579 backcross rats localized the swh locus to a .35-cM region between D17Rat131 and D17Rat50 in the distal end of rat Chr 17. The swh locus spanned the 3.7-Mb genomic region where 24 genes have been mapped and corresponded to the centromere region of the mouse Chr 2 or the region of the human Chr 10p11.1-p14. None of the genes or loci described in mouse or human hair and skin diseases mapped to these regions. These findings suggest that the rat swh is a novel mutation associated with impaired development of the skin appendages, such as hair follicles, sebaceous glands, and mammary glands, and will provide an experimental model to clarify a gene and mechanisms for development of skin appendages.     

Persistence of gene expression changes in stomach mucosae induced by short-term N-methyl-N'-nitro-N-nitrosoguanidine treatment and their presence in stomach cancers

Cancers induced by different carcinogens show distinct expression profiles. In addition to the specific alterations of tumor-related genes induced by specific carcinogens, it is possible that some initial responses induced by a carcinogen could persist for long periods and are consistently present in the cancers induced. We have analyzed the initial responses in the rat pyloric mucosae after treatment for 2 weeks with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Gene expression was monitored 1 day, 2 weeks and 4 weeks after MNNG treatment by oligonucleotide microarray analysis. Of the differentially expressed genes showing greater than three-fold difference 1 day after MNNG treatment, 143 and 26 genes were up- and down-regulated, respectively, in MNNG-induced stomach cancers. Among these genes, 25 and 6 genes were up- and down-regulated, respectively, in the histologically normal pyloric mucosae, even 4 weeks after cessation of MNNG treatment. Among the up-regulated genes, many genes involved in tissue remodeling (Spi15, Serpine1 and Fst) and cellular growth (Bdnf, Ros1 and Fgf10) were present. The six down-regulated genes included TGF-beta-inducible early growth response gene. These findings demonstrate that some expression changes induced by MNNG persist for a prolonged period and are present in cancers. Persistent expression changes are considered to be important for prediction of past carcinogen exposure, and could provide a molecular environment favorable for malignant transformation.     

Anti‐infectious activity of synbiotics in a novel mouse model of methicillin‐resistant Staphylococcus aureus infection

The anti‐infectious activity of synbiotics against methicillin‐resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 10⁸ CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5‐fluorouracil (5‐FU) was injected intraperitoneally on day 7 after the infection. A dose of 10⁸ CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 10⁸ CFU/g of intestinal contents after oral MRSA infection and the subsequent 5‐FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.     

Probiotic Lactobacillus casei activates innate immunity via NF-kappaB and p38 MAP kinase signaling pathways

Probiotic bacteria are microorganisms that benefit the host through improvement of the balance of intestinal microflora and possibly by augmentation of host defense systems. We examined the mechanisms for the up-regulation of innate immune responses by a probiotic Lactobacillus casei ATCC27139, in vivo. Using mouse models of systemic Listeria monocytogenes infection and MethA fibrosarcoma tumorigenesis in combination with BALB/c and SCID mice, we found that parenteral administration of L. casei ATCC27139 confers a protective effect against L. monocytogenes infection and anti-tumor activity against MethA fibrosarcoma by activation of innate immunity, while L. casei ATCC27139-J1R strains, which are J1 phage-resistant strains that have been selected from MNNG-treated clones, lacked these activities. Substantial differences between ATCC27139 and ATCC27139-J1R strains were observed in the capacity to induce innate cytokines such as TNF-alpha, IL-12, IL-18, and IFN-gamma, and pathogen-associated molecular pattern receptors, TLR2 and Nod2, by spleen cells. In addition, although phosphorylation of NF-kappaB p65 in spleen was equally enhanced in the ATCC27139- and the ATCC27139-J1R-treated groups, phosphorylation of both p38 MAPK and MAPKAPK-2 was significantly induced only by ATCC27139. Furthermore, inhibitors of NF-kappaB (sulfasalazine) and p38 MAPK (SB203580) significantly reduced cytokine production by the spleen cells of the mice treated with L. casei ATCC27139, suggesting that both NF-kappaB and p38 MAPK signaling pathways play important roles in the augmentation of innate immunity by the probiotic L. casei.     

Identification of the C=O stretching vibrations of FMN and peptide backbone by 13C-labeling of the LOV2 domain of Adiantum phytochrome3

Phototropin, a blue-light photoreceptor in plants, has two FMN-binding domains named LOV1 and LOV2. We previously observed temperature-dependent FTIR spectral changes in the C=O stretching region (amide-I vibrational region of the peptide backbone) for the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2), suggesting progressive structural changes in the protein moiety (Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191). Because FMN also possesses two C=O groups, in this article, we aimed at assigning C=O stretching vibrations of the FMN and protein by using 13C-labeling. We assigned the C(4)=O and C(2)=O stretching vibrations of FMN by using [4,10a-13C2] and [2-13C] FMNs, respectively, whereas C=O stretching vibrations of amide-I were assigned by using 13C-labeling of protein. We found that both C(4)=O and C(2)=O stretching vibrations shift to higher frequencies upon the formation of S390 at 77-295 K, suggesting that the hydrogen bonds of the C=O groups are weakened by adduct formation. Adduct formation presumably relocates the FMN chromophore apart from its hydrogen-bonding donors. Temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands. The hydrogen bond of the peptide backbone in the loop region is weakened upon S390 formation at low temperatures, while being strengthened at room temperature. The hydrogen bond of the peptide backbone in the alpha-helix is weakened regardless of temperature. On the other hand, structural perturbation of the beta-sheet is observed only at room temperature, where the hydrogen bond is strengthened. Light-signal transduction by phy3-LOV2 must be achieved by the progressive protein structural changes initiated by the adduct formation of the FMN.     

Vibrio cholerae/mimicus in fecal microbiota of healthy children in a cholera endemic urban slum setting in Kolkata,India

During a double‐blind, randomized, placebo‐controlled probiotic trial among 3758 children residing in an urban slum in Kolkata, India, Vibrio cholerae/mimicus was detected in fecal microbiota of healthy children. The importance of this finding in the local, regional and global transmission of cholera is discussed.