Characterization of the outermembrane proteins of Vibrio cholerae expressed in in vivo culture

Two outer membrane proteins (Omps) of Vibrio cholerae O1, expressed in the intestine (in vivo) but not in culture media (in vitro), were investigated. The molecular masses of those proteins were 116 kDa and 15 kDa, and they were not associated with iron-regulated proteins. Convalescent cholera patients' sera reacted with the 15 kDa protein but not with the 116 kDa protein. The N-terminal amino acid sequence of the 15 kDa protein was homologous to V. cholerae OmpT. Anti-serum to the 15 kDa protein caused agglutination of the organisms grown in the intestine, but not the organisms in in vitro culture. The anti-serum was bactericidal, but it did not inhibit the adhesion of the organisms to the intestine and HEp-2 cells. These findings suggest the possibility that the 15 kDa protein could be involved in post-infection immunity.     

The first isolation of Nocardia veterana from a human mycetoma

The clinical isolates from biopsy specimen human subcutaneous nodule developed orange-colored and wrinkled colonies on Sabouraud's dextrose agar at 24 C for 2 weeks. The isolates were aerobic and gram-positive. The bacteria were rod-shaped to coccoid and 1 x 5 microm in size. The assimilation tests revealed that the clinical isolate was identical to a reference strain of Nocardia veterana. A nucleotide sequence analysis of the 16S ribosomal DNA from the isolate and a reference strain of N. veterana showed 99.8% similarity. All data are consistent with the conclusion that the isolate in this human case of mycetoma is N. veterana.     

Inhibition of the expression of alpha-smooth muscle actin in human hepatic stellate cell line,LI90, by a selective cyclooxygenase 2 inhibitor,NS-398

Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS-398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC.     

Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice

Plasminogen activator inhibitor-1 (PAI-1)-deficient transgenic mice have improved survival and less fibrosis after intratracheal bleomycin instillation. We hypothesize that PAI-1 deficiency limits scarring through unopposed plasminogen activation. If this is indeed true, then we would expect increased urokinase-type plasminogen activator (uPA) expression to result in a similar reduction in scarring and improvement in mortality. To test our hypothesis, using the tetracycline gene regulatory system, we have generated a transgenic mouse model with the features of inducible, lung-specific uPA production. After doxycycline administration, these transgenic animals expressed increased levels of uPA in their bronchoalveolar lavage (BAL) fluid that accelerated intrapulmonary fibrin clearance. Importantly, this increased plasminogen activator production led to a reduction in both lung collagen accumulation and mortality after bleomycin-induced injury. These results suggest that PAI-1 deficiency does protect against the effects of bleomycin-induced lung injury through unopposed plasmin generation. By allowing the manipulation of plasminogen activation at different phases of the fibrotic process, this model will serve as a powerful tool in further investigations into the pathogenesis of pulmonary fibrosis.     

Female spawning strategy in Rhinogobius sp. OR: how do females deposit their eggs in the nest?

 We tried to elucidate how females of a paternal nest brooding goby Rhinogohius sp. OR deposit their eggs in a nest, using a marking technique for live eggs under laboratory conditions in which male somatic condition, nest space, and mating pattern (monogamous or bigamous) were controlled. Whether females rejected mating was independent of either male quality, such as body size and somatic condition, or nest space. In a situation in which two females were allowed to spawn sequentially with a male, however, females rejected mating at a higher rate when they were the first to spawn than when they were the second to spawn; this is because eggs from first females were more vulnerable to cannibalism by parental males and second females. Even when nest space was limited and thus was occupied by eggs from the first females, second females could deposit all their eggs in the nest by using the minute interspace of existing eggs. In the presence of the parental male, such a female seemed less likely to suffer a cost from increased egg mortality due to crowdedness, still holding the advantage of being the second spawner. Finally, we extrapolated the field breeding ecology of this fish from the laboratory data. It was suggested that a single monolayer brood of the same age usually consisted of eggs from multiple females and thus the mating pattern would be more polygynous than previously expected. Received: March 6, 2002 / Revised: July 11, 2002 / Accepted: August 14, 2002 RID="*" ID="*" Present address: Center for Marine Environmental Studies, Ehime University, 3 Bunkyo-cho, Matsuyama 790-8577, Japan (e-mail: nokuda@sci.ehime-u.ac.jp) Acknowledgments I am grateful to S. Sone and D. Takahashi for giving us useful information and to M. Inoue and H. Miyatake for their field assistance. This study was financially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Correspondence to:Noboru Okuda     

Molecular characterization of sessile serrated adenoma/polyps with dysplasia/carcinoma based on immunohistochemistry,next-generation sequencing,and microsatellite instability testing: a case series study

Background

Colorectal sessile serrated adenoma/polyps (SSA/Ps) are considered early precursor lesions in the serrated neoplasia pathway. Recent studies have shown associations of SSA/Ps with lost MLH1 expression, a CpG island methylator phenotype, and BRAF mutations. However, the molecular biological features of SSA/Ps with early neoplastic progression have not yet been fully elucidated, owing to the rarity of cases of SSA/P with advanced histology such as cytologic dysplasia or invasive carcinoma. In this study, we aimed to elucidate the molecular biological features of SSA/Ps with dysplasia/carcinoma, representing relatively early stages of the serrated neoplasia pathway.

Methods

We performed immunostaining for β-catenin, MLH1, and mucins (e.g., MUC2, MUC5AC, MUC6, and CD10); targeted next-generation sequencing; and microsatellite instability (MSI) testing in 8 SSA/P lesions comprised of 4 SSA/Ps with high-grade dysplasia and 4 SSA/Ps with submucosal carcinoma.

Results

Lost MLH1 expression was found in 5 cases. All lesions studied were positive for nuclear β-catenin expression. Regarding phenotypic mucin expression, all lesions were positive for MUC2, but negative for CD10. MUC5AC and MUC6 positivity was observed in 7 cases. Genetically, the most frequently mutated gene was BRAF (7 cases), and other mutations were detected in FBXW7 (3 cases); TP53 (2 cases), and KIT, PTEN, SMAD4, and SMARCB1 (1 case each). Furthermore, 4 of 8 lesions were MSI-high and the remaining 4 lesions were microsatellite-stable (MSS). Interestingly, all 4 MSI-high lesions displayed MLH1 loss, 3 of which harbored a FBXW7 mutation, but not a TP53 mutation. However, 2 MSS lesions harbored a TP53 mutation, although none harbored a FBXW7 mutation.

Conclusions

SSA/Ps with dysplasia/carcinoma frequently harbored BRAF mutations. Activation of the WNT/β-catenin signaling pathway may facilitate the development of dysplasia in SSA/Ps and progression to carcinoma. Furthermore, our results suggested that these lesions might be associated with both MSI-high and MSS colorectal cancer, which might be distinguished by distinct molecular biological features such as lost MLH1 expression, FBXW7 mutations, and TP53 mutations.

     

Isolation and Identification of L-Pipecolic Acid and Nicotinamide as Flower-Inducing Substances in Lemna

Flower-inducing factors in extracts of flowering Lemna gibbaG3 were investigated using Lemna paucicostata 151 as the bioassayplant. Fractions with flower-inducing activity were obtainedafter several purification steps. Two of the active substanceswere identified as L-pipecolic acid and nicotinamide by MS andNMR analyses. Both L-pipecolic acid and nicotinamide exhibited flower-inducingactivity in L. paucicostata 151 grown on one-tenth-strengthM medium containing benzyladenine, the former being ten timesas active as the latter. L-Pipecolic acid was active even at0.01 ppm (7.8 ? 10^–8 M). The effect of L-pipecolic acidon flowering strongly depended upon the presence of exogenouscytokinin. The coexistence of cytokinin seemed to be essentialfor L-pipecolic acid to exhibit flower-inducing activity. Incontrast, the effect of nicotinamide on flowering was basicallythe same as that of benzoic acid or nicotinic acid. (Received February 9, 1987; Accepted May 21, 1987)     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Concentrations ofent-kaurene and squalene in vegetative rice shoots

Ent-kaurene (ent-kaur-16-ene) and squalene were analyzed in extracts of the shoots of three cultivars of rice (Oryza sativa L.) of 14 and 28 days of age by gas chromatography-mass spectrometry (GS-MS) and GC-selected ion monitoring (GS-SIM).Ent-kaurene occurred at approximate concentrations of <1 to 13 ng/g f.w. in 14-day-old plants and 26 to 147 ng/g f.w. in 28-day-old plants. Shoots of the dwarf cultivar Waito-C contained much lessent-kaurene than the other two cultivars at both developmental stages. The level ofent-kaurene in the dwarf cultivar Tan-ginbozu was similar to that in the normal cultivar Nihonbare at 14 days but was lower than in Nihonbare at 28 days. Trace amounts ofent-isokaurene (ent-kaur-15-ene) were also found in the extracts of all three cultivars of shoots at 28 days. Squalene occurred in approximate concentrations from as low as 19 ng/g f.w. in 28-day-old Waito-C shoots to as much as 626 ng/g f.w. in 14-day-old Nihonbare shoots. Both Tan-ginbozu and Waito-C shoots contained less squalene than Nihonbare shoots at both developmental stages.     

Nucleotide sequence of the 16S-23S spacer region in the rrnA operon from a blue-green alga, Anacystis nidulans

Summary The nucleotide sequence of an entire spacer region between the 16S and 23S rRNA genes of the rrnA operon from a blue-green alga, Anacystis nidulans, has been determined. The spacer region is 545 base pairs long and encodes tRNA^fle and tRNA^Ala in the order of 16S rRNA-tRNA^fle-tRNA^Ala-23S rRNA. A striking feature is that the A. nidulans tRNA^fle gene contains no 3-CCA sequence while the tRNA^Ala gene does. These spacer tRNA genes show strong sequence homology with those of chloroplasts and bacteria.