Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan

The occurrence and characterization of toxigenic vibrios in surface water and sediment samples of the fresh water environment of the Ohta River were studied. The membrane filter, pad preenrichment technique, followed by the placement of membranes onto thiosulfate citrate-bile salt-sucrose agar, was used for the enumeration of total vibrios. Qualitative examination of pathogenic vibrios was also attempted. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples of the Ohta River and the Hiroshima coast. In the identification of 361 strains, 12 species of Vibrio and two species of Listonella were observed. Non-01 Vibrio cholerae was prevalent among the members of the genus Vibrio. Vibrio parahaemolyticus (serotype 04:K34), isolated in fresh water, is significant and suggests that some still unknown conditions promote the survival of these organisms in fresh water. An estimated 132 strains were hemolytic by a simple agar method, and further characterization revealed that 82% of the hemolytic vibrios (107 strains) produced various toxins. About 71% (93 strains) elaborated cytotoxin, 55% (72 strains) produced hemolysin, and 44% (58 strains) responded for both cytotoxin and hemolysin in the crude toxin extracts. All the non-01 V. cholerae showed cytotoxic activity, and the virulent strains of Vibrio fluvialis and Vibrio spp. showed cytotonic responses in RK-13 cells. Of 36 sediment samples tested, 10 harbored C. botulinum spores (28%) and were isolated invariably in all the regions of the Hiroshima coast and in the Ohta River, except the upper region of the Ohta River.     

Synthesis of peptide thioesters via an N–S acyl shift reaction under mild acidic conditions on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl auxiliary group

An efficient method of peptide thioester synthesis is described. The reaction is based on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl (Dmmb) auxiliary‐assisted N‐S acyl shift reaction after assembling a peptide chain by Fmoc‐solid phase peptide synthesis. The Dmmb‐assisted N‐S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S‐peptide with sodium 2‐mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on‐resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41‐mer peptide thioester, a phosphorylated peptide thioester and a 33‐mer peptide thioester containing a trimethylated lysine residue. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

NK cells eliminate Cryptococcus neoformans by potentiating the fungicidal activity of macrophages rather than by directly killing them upon stimulation with IL-12 and IL-18

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)-12 and IL-18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN-gamma and exerted marked cytotoxic activity against YAC-1 cells. On the other hand, NK cells significantly potentiated the nitric oxide-mediated cryptococcocidal activity of thioglycolate-elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL-12 and IL-18. The culture supernatants of NK cells stimulated with IL-12 and IL-18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN-gamma because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage-mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.     

NOD-Rag2null IL-2Rγnull Mice: An Alternative to NOG Mice for Generation of Humanized Mice

We have developed NOD-Rag2^null IL-2Rγ^null (NR2G) mice similar to NOD-scidIL-2Rγ^null (NOG) mice that are known as an excellent host to generate humanized mice. To evaluate the usefulness of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of human cells after human hematopoietic stem cell (HSC) transplantation were compared among NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate irradiation doses to expand the niche for human stem cells in the bone marrow were first determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG mice, respectively, were found to be appropriate. Next, 5 × 10⁴ human umbilical cord blood CD34⁺ cells were intravenously inoculated into irradiated adult or newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated that both NR2G and NOG mice showed high engraftment rates compared with NOD-scid mice, although NOG mice showed a slightly higher engraftment rate than that for NR2G mice. However, no difference was found in the human cell populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation experiments to adults and newborns of two immunodeficient mice also revealed that the HSC transplantation into newborn mice resulted in higher engraftment rate than those into adults. These results showed that NR2G mice could be used as an alternative host to NOG mice to generate humanized mice.     

Six1 is indispensable for production of functional progenitor cells during olfactory epithelial development

The rodent olfactory epithelium (OE) is a good model system for studying the principles of stem and progenitor cell biology, because of its capacity for continuous neurogenesis throughout life and relatively well-characterized neuronal lineage. The development of mouse OE is divided into two stages, early and established neurogenesis. In established neurogenesis, which starts at embryonic day (E) 12.5, sustentacular cells and olfactory receptor neurons (ORNs) are produced from apical and basal progenitors, respectively. We previously reported that Six1(-/-) shows a lack of mature ORNs throughout development and disorganization of OE after E12.5. However, the molecular bases for these defects have not been addressed. Here, we show that Six1 is expressed in both apical and basal progenitors. In Six1(-/-) mice, apical proliferating cells were absent and no morphologically identifiable sustentacular cells were observed. Consistently, the expression of Notch2 and Jagged1 in the apical layer was absent in Six1(-/-) mice. On the other hand, basal proliferating cells were observed in Six1(-/-) animals, but the expression of Ngn1, NeuroD, Notch1, and Jagged2 in the basal layer was absent. The expression of Mash1, the determination gene for ORNs, and Hes genes was enhanced in Six1(-/-) mice. The present findings suggest that Six1 regulates production of functional apical and basal progenitors during OE development, through the regulation of various genes, such as neuronal basic helix-loop-helix (bHLH), neuronal repressor bHLH, and genes involved in the Notch signaling pathway.     

Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells

BACKGROUND: RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells. METHODS: We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors. RESULTS: As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five. CONCLUSION: The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi.     

Location of sialoglycoconjugates containing the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups in the rat hippocampus and the effect of aging on their expression.

The histochemical distribution of sialoglycoconjugates in the CA1 region in the hippocampus formation of 9-week-old rats and 30-month-old rats was examined using electron microscopy in combination with two lectins, Maackia amurensis lectin, specific for Sia(alpha)2-3Gal, and Sambucus sieboldiana agglutinin, specific for Sia(alpha)2-6Gal. Each lectin stained the plasma membranes of pyramidal cells, indicating that the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups were expressed on their plasma membranes. These lectins also bound to synapses in the stratum lacunosum molecular. The staining intensity of the lectins in the synapses in these layers was downregulated in the 30-month-old rats. These results indicated that both the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups are expressed on these synapses and that the expression of these sialyl linkages decreases in the aged brain     

Low-Frequency IL23R Coding Variant Associated with Crohn’s Disease Susceptibility in Japanese Subjects Identified by Personal Genomics Analysis

Background

The common disease-common variant hypothesis is insufficient to explain the complexities of Crohn’s disease (CD) genetics; therefore, rare variants are expected to be important in the disease. We explored rare variants associated with susceptibility to CD in Japanese individuals by personal genomic analysis.

Methods

Two-step analyses were performed. The first step was a trio analysis with whole-exome sequence (WES) analysis and the second was a follow-up case-control association study. The WES analysis pipeline comprised Burrows-Wheeler Aligner, Picard, Genome Analysis Toolkit, and SAMTOOLS. Single nucleotide variants (SNVs)/indels were annotated and filtered by using programs implemented in ANNOVAR in combination with identity-by-descent (IBD), subsequently were subjected to the linkage based, and de novo based strategies. Finally, we conducted an association study that included 176 unrelated subjects with CD and 358 healthy control subjects.

Results

In family members, 234,067–297,523 SNVs/indels were detected and they were educed to 106–146 by annotation based filtering. Fifty-four CD variants common to both individuals of the affected sib pair were identified. The linkage based strategy detected five candidate variants whereas the de novo based strategy identified no variants. Consequently, five candidates were analyzed in the case-control association study. CD showed a significant association with one variant in exon 4 of IL23R, G149R [rs76418789, P = 3.9E-5, odds ratio (OR) 0.21, 95% confidence interval (CI) 0.09–0.47 for the dominant model (AA + AG versus GG), and P = 7.3E-5, OR 0.21, 95% CI 0.10–0.48 for AG versus GG, and P = 7.2E-5, OR 0.23, 95% CI 0.10–0.50 for the allele model].

Conclusions

The present study, using personal genomics analysis of a small CD pedigree, is the first to show that the low-frequency non-synonymous variant of IL23R, rs76418789, protects against CD development in Japanese subjects.     

Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE)

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.