Seasonality of coastal upwelling trends in the Mauritania-Senegalese region under RCP8.5 climate change scenario.

The Mauritania-Senegalese upwelling region (MSUR), the southernmost region of the Canary current upwelling system, is well-known for its coastal productivity and the key role it plays in enriching the oligotrophic open ocean through the offshore transport of the upwelled coastal waters. The great ecological and socio-economic importance makes it necessary to evaluate the impact of climate change on this region. Hence, our main objective is to examine the climate change signal over the MSUR with a high resolution regional climate system model (RCSM) forced by the Earth system model MPI-ESM-LR under RCP8.5 scenario. This RCSM has a regional atmosphere model (REMO) coupled to a global ocean model (MPIOM) with high-resolution in the MSUR, which allows us to evaluate the wind pattern, the ocean stratification, as well as the upwelling source water depth, while maintaining an ocean global domain. Under RCP8.5 scenario, our results show that the upwelling favourable winds of the northern MSUR are year-round intensified, while the southern MSUR presents a strengthening in winter and a weakening in March-April. Along with changes in the wind pattern, we found increased ocean stratification in the spring months. In those months southern MSUR presents a shallowing of the upwelling source water depth associated to changes in both mechanisms. However, in winter the whole MSUR shows a deepening of the upwelling source water depth due to the intensification of the upwelling favourable winds, with the increased ocean stratification playing a secondary role. Our results demonstrate the need to evaluate the future evolution of coastal upwelling systems taking into account their latitudinal and seasonal variability and the joint contribution of both mechanisms.

Modelling the invasion of human small intestinal epithelial-like cells by Salmonella enterica Typhimurium and Listeria monocytogenes using Bayesian inference.

To develop a mechanistic bacterial dose-response model, based on the concept of Key Events Dose-Response Framework (KEDRF), this study aimed to investigate the invasion of intestinal model cells (Caco-2) by Salmonella Typhimurium and Listeria monocytogenes and described the behaviour of both pathogens as a mathematical model using Bayesian inference. Monolayer-cultured Caco-2 cells (approximately 105 cells) were co-cultured with various concentrations (103 -107 colony forming unit [CFU] ml-1 ) of Salm. Typhimurium and L. monocytogenes for up to 9 h to investigate the invasion of the pathogens into the Caco-2 cells. While an exposure of ≥103 CFU ml-1 of Salm. Typhimurium initiated the invasion of Caco-2 cells within 3 h, much less exposure (102 CFU ml-1 ) of L. monocytogenes was sufficient for invasion within the same period. Furthermore, while the maximum number of invading Salm. Typhimurium cells reached by approximately 103 CFU cm-2 for 6-h exposure, the invading maximum numbers of L. monocytogenes cells increased by approximately 106 CFU cm-2 for the same exposure period. The invasion kinetics of both the pathogens was successfully described as an asymptotic exponential mathematical model using Bayesian inference. The developed pathogen invasion model allowed the estimation of probability of Salm. Typhimurium and L. monocytogenes infection, based on the physiological natures of digestion process, which was comparable to the published dose-response relationship. The invasion models developed in the present study will play a key role in the development of an alternative pathogen dose-response model based on KEDRF concept.

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress.

AIMS: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. METHODS AND RESULTS: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55-70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE™ system to measure net K(+) fluxes. No difference in K(+) fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K(+) loss from the heat-treated E.coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K(+) flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K(+) flux disruption was not clearly observed until 68°C was reached. CONCLUSIONS: No changes in net K(+) flux from heat-stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K(+) fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two-stage process enabled evaluation of the lethality of heat-treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture-dependent test offers an alternative system to culture-based methods.

Effect of specific antigen stimulation on intraepithelial lymphocyte migration to small intestinal mucosa.

Migration of intraepithelial lymphocytes (IELs) into intestinal epithelium is not yet well understood. We established an IEL-cell line from ovalbumin (OVA) 23-3 transgenic (Tg) mice and investigated the effect of antigen stimulation on the dynamic process of IEL migration into small intestinal mucosa. The cell line was a T cell receptor (TCR) alphabeta(+) CD4(+) CD8(-) phenotype, expressing alphaEbeta7 integrin in 90% of cells. Under intravital microscopy, the lined IELs adhered selectively to the microvessels of the intestinal villus tip of the Tg mice. The accumulation of IELs was significantly inhibited by an antibody against beta7-integrin and MAdCAM-1. When IELs were stimulated with OVA, the accumulation was attenuated compared to that of resting cells, with decreased expression of alphaEbeta7 integrin. In Tg mice fed with OVA, the number of IELs which migrated in the villus mucosa was significantly smaller than in the non-fed controls. The preferential migratory capacity of IELs to villus mucosa may be altered by specific antigen stimulations.

Intercellular cell adhesion molecule-1 regulates lymphocyte movement into intestinal microlymphatics of rat Peyer's patches.

The objective of this study was to determine whether specific adhesion molecules modulate lymphocyte movement from Peyer's patches into intestinal microlymphatics. The fluorochrome acridine orange was injected via a micropipette into Peyer's patches to fill lymphatics. The flux of labeled lymphocytes into intestinal microlymphatics was monitored with intravital fluorescence microscopy. The lymphatic microvessels in the perifollicular area of Peyer's patches were filled with lymphocytes, most of which remained within the lymphatics. Some lymphocytes became detached and were drained into intestinal lymph. Administration of antibodies directed against ICAM-1 significantly increased lymphocyte flux into interfollicular lymphatics. The immunohistochemical study showed intense ICAM-1 expression on the lymphocytes densely packed in the lymphatics surrounding follicles in Peyer's patches. A large number of lymphocytes are normally sequestered in the lymphatic network of Peyer's patches. This sequestration of lymphocytes is largely mediated by ICAM-1-dependent cell-cell interactions.

In situ demonstration of intraepithelial lymphocyte adhesion to villus microvessels of the small intestine.

The recirculation of lymphocytes through the intestinal mucosa is important for specific immune defense, but the origin and differentiation of intraepithelial lymphocytes (IEL) are not fully understood. The present study therefore used intravital microscopy to investigate the migration of IEL to the villus mucosa and Peyer's patches of the small intestine. IEL were separated from inverted murine small intestine and mesenteric lymph node (MLN) T cells were also isolated. The adhesion of fluorescence-labeled lymphocytes to postcapillary venules (PCV) of Peyer's patches and arcade microvessels of small intestinal villi was observed after injection. In some experiments, the effect of antibodies against adhesion molecules on cell kinetics were investigated. IEL time-dependently accumulated in villus microvessels of the small intestine, whereas few MLN cells did. Few IEL adhered to the PCV of Peyer's patches. IEL were shown to express alpha(E)beta(7)-integrin but not L-selectin. The accumulation of IEL in villus archade was significantly inhibited by antibody against beta(7)-integrin or mucosal addressin cell adhesion molecules (MAdCAM)-1, but not by alpha(E)-integrin. The combined blocking of beta(7)-integrin and MAdCAM-1 further attenuated the sticking of IEL in this area, although it did not entirely block the IEL adherence. The adherence of CD4(+) or TCRalphabeta IEL to villus microvessels was significantly greater than that of CD4(-) or TCRgammadelta IEL. It was demonstrated in situ for the first time that IEL adhered selectively to the villus microvessels of the small intestine partly via beta(7) and MAdCAM-1.

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Decontamination of lettuce using acidic electrolyzed water.

The disinfectant effect of acidic electrolyzed water (AcEW), ozonated water, and sodium hypochlorite (NaOCl) solution on lettuce was examined. AcEW (pH 2.6; oxidation reduction potential, 1140 mV; 30 ppm of available chlorine) and NaOCl solution (150 ppm of available chlorine) reduced viable aerobes in lettuce by 2 log CFU/g within 10 min. For lettuce washed in alkaline electrolyzed water (AIEW) for 1 min and then disinfected in AcEW for 1 min, viable aerobes were reduced by 2 log CFU/g. On the other hand, ozonated water containing 5 ppm of ozone reduced viable aerobes in lettuce 1.5 log CFU/g within 10 min. It was discovered that AcEW showed a higher disinfectant effect than did ozonated water significantly at P < 0.05. It was confirmed by swabbing test that AcEW, ozonated water, and NaOCI solution removed aerobic bacteria, coliform bacteria, molds, and yeasts on the surface of lettuce. Therefore, residual microorganisms after the decontamination of lettuce were either in the inside of the cellular tissue, such as the stomata, or making biofilm on the surface of lettuce. Biofilms were observed by a scanning electron microscope on the surface of the lettuce treated with AcEW. Moreover, it was shown that the spores of bacteria on the surface were not removed by any treatment in this study. However, it was also observed that the surface structure of lettuce was not damaged by any treatment in this study. Thus, the use of AcEW for decontamination of fresh lettuce was suggested to be an effective means of controlling microorganisms.

Truncated mutant B subunit for factor XIII causes its deficiency due to impaired intracellular transportation.

Two Japanese patients were newly diagnosed as having B subunit (XIIIB) deficiency of factor XIII (former type I deficiency). Both patients have a previously described one-base deletion at the boundary between intron A/exon II in the XIIIB gene, heterozygously or homozygously. A founder effect was proposed for this mutation because 3 unrelated patients with XIIIB deficiency also share 2 3'-polymorphisms. In one patient heterozygous for the above mutation, a novel mutation was also identified: a deletion of guanosine in exon IX (delG) of the XIIIB gene. To understand the molecular and cellular pathology of the delG mutation, expression studies were performed using a cultured mammalian cell line. Pulse-chase experiments showed that a resultant truncated XIIIB remained inside the cells and could not be secreted into the culture medium. Furthermore, immunocytochemical examinations by epifluorescence, confocal, and electron microscopes indicated impaired intracellular transportation of the truncated XIIIB from the endoplasmic reticulum to the Golgi apparatus. No mutations in the gene for the A subunit (XIIIA) were identified in this patient. Therefore, secretion of the truncated XIIIB must also be impaired in vivo, leading to a secondary XIIIA deficiency. These results support a previous conclusion that genetic defects of XIIIB are the basis for the former type I factor XIII deficiency.

Intravital demonstration of modulation of T lymphocyte migration by CINC/gro in rat Peyer's patches.

BACKGROUND/AIMS: Cytokine-induced neutrophil chemoattractant (CINC/gro), a member of interleukin-8 family, was found as a potent chemotactic factor for rat neutrophils. Although several chemokines have been shown to be potent regulators of T cell chemotaxis in vitro, the potential role of chemokines in T-cell migration in gut-associated lymphoid tissues has not been investigated in vivo. In the present study, the effects of CINC/gro on T-lymphocyte migration were examined in rat Peyer's patches. METHODS: T lymphocytes collected from intestinal lymph of rats were fluorescence-labeled and injected into the jugular vein. Peyer's patches of the recipient rats were observed with intravital fluorescence microscopy and the effects of CINC/gro infusion was investigated. Lymphocyte flux in mesenteric collecting lymphatics was also observed. RESULTS: In vivo infusion of CINC/gro significantly attenuated the initial lymphocyte interaction with postcapillary venules of Peyer's patches. However, once these lymphocytes adhered to venules, CINC/gro treatment significantly accelerated the transendothelial migration of T lymphocytes and they also significantly increased their subsequent flux in collecting lymphatics. CONCLUSION: There is a possibility that CINC/gro could modulate the characteristics of T lymphocyte homing in the inflammatory sites of gut.

Prediction of microbial growth in fresh-cut vegetables treated with acidic electrolyzed water during storage under various temperature conditions.

Effects of storage temperature (1, 5, and 10 degrees C) on growth of microbial populations (total aerobic bacteria, coliform bacteria, Bacillus cereus, and psychrotrophic bacteria) on acidic electrolyzed water (AcEW)-treated fresh-cut lettuce and cabbage were determined. A modified Gompertz function was used to describe the kinetics of microbial growth. Growth data were analyzed using regression analysis to generate "best-fit" modified Gompertz equations, which were subsequently used to calculate lag time, exponential growth rate, and generation time. The data indicated that the growth kinetics of each bacterium were dependent on storage temperature, except at 1 degrees C storage. At 1 degrees C storage, no increases were observed in bacterial populations. Treatment of vegetables with AcEW produced a decrease in initial microbial populations. However, subsequent growth rates were higher than on nontreated vegetables. The recovery time required by the reduced microbial population to reach the initial (treated with tap water [TW]) population was also determined in this study, with the recovery time of the microbial population at 10 degrees C being <3 days. The benefits of reducing the initial microbial populations on fresh-cut vegetables were greatly affected by storage temperature. Results from this study could be used to predict microbial quality of fresh-cut lettuce and cabbage throughout their distribution.

Characterization of anti-irradiation-denatured ovalbumin monoclonal antibodies. Immunochemical and structural analysis of irradiation-denatured ovalbumin.

Five monoclonal antibodies (OVA-01, -02, -03, -04, -06) produced against irradiated ovalbumin were investigated in relation to the conformational change in the ovalbumin molecule induced by irradiation with Cobalt-60 gamma-rays. Four antibodies (OVA-01, -02, -04, -06) recognized both native and irradiated ovalbumin, but OVA-03 reacted only with irradiated ovalbumin. These antibodies were classified by modified competitive ELISA, and their K(d) values were determined by the Klotz equation. Epitope analyses were also performed on OVA-03 using CNBr-cleaved peptide fragments from ovalbumin, and it was confirmed that OVA-03 bound to the fragment corresponding to residues Val173-Met196 of the ovalbumin molecule that consists of internal beta-sheet strand 3A and helix F1 containing one open turn. These results demonstrate that dramatic conformational changes in proteins can be induced or that some tertiary or secondary structures can be broken down by gamma-ray irradiation, producing new antigenic sites.

E-cadherin expression in skin tumors using an AMeX immunohistostaining method.

The AMeX (acetone-methylbenzoate-xylene) method results in good preservation of tissue and morphological details, almost equivalent to that of routinely processed formalin-fixed and paraffin-embedded tissue specimens, and of antigenicity equivalent to that of fresh frozen tissue specimens. It has been reported that the expression of the cell-cell adhesion molecule E-cadherin is often decreased in some types of carcinomas. A decrease in E-cadherin expression is associated with the invasive or metastatic potential of tumor cells. We immunohistochemically examined the expression of E-cadherin with anti-E-cadherin monoclonal antibody in various skin tumors (25 basal cell carcinomas, 11 squamous cell carcinomas, 9 keratoacanthomas, and 11 Bowen's disease) using the AMeX method and found that this method preserved antigenicity well without pretreatment. E-cadherin expression was decreased in 18.2% of squamous cell carcinomas and 33.3% of keratoacanthomas. On the other hand, it was preserved in almost all Bowen's disease and basal cell carcinomas. From the results of our study, we suggest that Bowen's disease and basal cell carcinoma do not have much metastatic potential due to retention of high levels of E-cadherin expression. We hope to apply the AMeX method to other immunohistochemical examinations because this is a very useful staining method.

Squamous cell carcinoma arising in chronic perianal pyoderma a case report and review of Japanese literature.

We report a rare case of squamous cell carcinoma developing from fistules of chronic perianal pyoderma in a 49-year-old Japanese man. He first noticed an abscess and nodule on his buttocks and perianal area 21 year previously (at the age of 28); the fistules formed later. These fistules were surgically removed, and an artificial anus was constructed 14 years ago (at the age of 35) in our hospital, when a histopathological examination revealed no malignant changes. However, he was recently admitted to our hospital with arterial bleeding from the ulcer of the buttock. On admission, the histological diagnosis of the ulcer was well differentiated squamous cell carcinoma. Wide local excision of the ulcer and scar tissue, including the sacrum, was performed. The defect was covered with a left latissimus dorsi flap and skin graft. He received radiation therapy after the operation. However, he died of cachexia and pneumonia. This case indicated that the CPP would better have been treated with wide excision before the development of SCC. Therefore, we recommend careful follow-up of patients affected by CPP and repeated biopsies of the lesion, particularly when the condition is severe, longstanding, and extensive. We discussed the term "CPP" and reviewed 22 cases of SCC arising in CPP reported in the Japanese literature.

Altered migration of gut-derived T lymphocytes after activation with concanavalin A.

Although activation of lymphocytes is known to be associated with profound changes in homing behavior, it remains unclear how activation alters migration of gut-derived lymphocytes in lymphoid and nonlymphoid organs. The objectives of this study were 1) to compare migration of naive and concanavalin A (ConA)-activated T lymphocytes into the gut mucosa, spleen, and liver and 2) to define the role of specific adhesion molecules in this homing process. Fluorescently labeled T lymphocytes collected from rat intestinal lymph were injected into the jugular vein, and the kinetics of appearance of the infused lymphocytes were monitored in ileal Peyer's patches, spleen, and liver. The migration of naive and ConA-activated T lymphocytes into microvessels were compared using an intravital microscope. ConA stimulation significantly increased the rolling velocity of T lymphocytes in postcapillary venules of Peyer's patches, and ConA-stimulated lymphocytes exhibited a loss of the selective adherence properties in Peyer's patches that is normally observed with naive T cells. ConA activation also suppressed the accumulation of T cells in the spleen. On the other hand, the adherence of T cells to hepatic sinusoidal endothelium was significantly increased after ConA activation, especially in the periportal area, and this increase was attenuated by an anti-intercellular adhesion molecule (ICAM)-1 antibody. Flow cytometry analysis revealed a decline in L-selectin expression and an increase in CD11a expression and ICAM-1 on the surface of ConA-treated T cells. In conclusion, activation of gut-derived T lymphocytes with ConA significantly alters their migration path, with a diminished localization to Peyer's patches and spleen and a preferential accumulation in hepatic sinusoids. This altered migration pattern likely results from changes in the expression of leukocyte adhesion molecules such as L-selectin and CD11a.

An immunohistochemical study of E-cadherin expression in human squamous cell carcinoma of the skin: relationship between decreased expression of E-cadherin in the primary lesion and regional lymph node metastasis.

E-cadherin is a Ca(2+)-dependent, intercellular adhesion molecule that is specifically expressed in epithelial tissues and is essential for maintaining intercellular connections. It has been reported that E-cadherin expression of tumor cells is often decreased in some types of metastasizing carcinomas as compared with those without metastasis. We immunohistochemically examined the expression of E-cadherin with anti-E-cadherin monoclonal antibody and compared primary lesions of human squamous cell carcinoma of the skin (SCC) with regional lymph node metastasis to those without regional lymph node metastasis. Tumor samples from fifty-five cases of SCC (32 cases of SCC without metastasis and 23 cases with metastasis) were formalin-fixed, paraffin-embedded, and examined. E-cadherin was reduced or absent in 39 (70.9%) out of 55 cases of SCC, and in 21 (91.3%) of 23 cases with regional lymph node metastasis. Our results suggest that the decreased expression of E-cadherin in the primary lesion is correlated with regional lymph node metastasis in SCC and that it is more frequently correlated with well-differentiated than with poorly differentiated SCC. E-cadherin may be useful as a marker for metastatic potential in well-differentiated SCC.

Factors governing the activity in vivo of ribozymes transcribed by RNA polymerase III.

In order to determine the parameters that govern the activity of a ribozyme in vivo, we made a systematic analysis of chimeric tRNAVal ribozymes by measuring their cleavage activities in vitro as well as the steady-state levels of transcripts, the half-lives of transcribed tRNAVal ribozymes, and their activities in both HeLa and H9 cells. These analyses were conducted by the use of transient expression systems in HeLa cells and stable transformants that express ribozymes. Localization of transcripts appeared to be determined by the higher-order structure of each transcribed tRNAVal ribozyme. Since colocalization of the ribozyme with its target RNA is important for strong activity of the ribozyme in vivo, the best system for tRNA-based expression seems to be one in which the structure of the transcript is different from that of the natural tRNA precursor so that processing of the tRNAVal ribozyme can be avoided. At the same time, the structure of the transcript must be similar enough to allow recognition, probably by an export receptor, so that the transcript can be exported to the cytoplasm to ensure colocalization with its target. In the case of several tRNAVal ribozymes that we constructed, inspection of computer-predicted secondary structures enabled us to control the export of transcripts. We found that only a ribozyme that was transcribed at a high level and that had a sufficiently long half-life, within cells, had significant activity when used to withstand a challenge by human immunodeficiency virus type 1.

[A case of multiple early gastric cancer showing complete response to UFT-E after endoscopic mucosal resection].

A 78-year-old male underwent endoscopic mucosal resection for early gastric cancer (type II a) in the anterior wall of antrum. Follow-up endoscopy showed another early gastric cancer (type II c) in the posterior wall of cardia. He refused an operation. UFT-E 300 mg/day has been administered daily since then. After 6 months, a complete tumor response was observed. The patient has been in good health without any sign of relapse for 14 months after achievement of the complete response.

Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

[Mucosal immune system of the intestine].

Gut-associated lymphoid tissue is the major inductive site of mucosal immune system, functionally independent of the systemic immune system. Particulate antigens are mainly uptaken from M cell of Peyer's patches, inducing IgA production in the intestinal mucosa. Lymphocytes are continuously recirculating through the intestinal mucosa to facilitate intestinal immune response. Dysregulation of lymphocyte migration and cytokine imbalance in the intestinal mucosa may be largely involved in the pathogenesis of inflammatory bowel diseases including intestinal allergy and Crohn's disease. There is also a possibility that dietary components especially long chain fatty acid could influence immune cell function of the intestinal mucosa. Because dietary components are closely associated with immunological function of intestinal mucosa, the importance of dietary manipulation for the management of inflammatory bowel diseases should be concerned.