ABSTRACT:The absorption characteristics of temocapril were investigated using Caco-2 cells, and the esterases expressed in Caco-2 cells were identified. Temocapril was almost completely hydrolyzed to temocaprilat during transport across Caco-2 cells. Hydrolysis experiments of temocapril in Caco-2 cell 9000g supernatant (S9) and brush-border membrane vesicles showed that temocapril was mainly hydrolyzed within the cells after uptake, after which the temocaprilat formed was transported to both the apical and basolateral surfaces. In native polyacrylamide gel electrophoresis by detection of hydrolase activity for 1-naphthylbutyrate, Caco-2 cell S9 showed a band with high esterase activity and another band with extremely low activity. The proteins in the major and minor bands were identified as carboxylesterase-1 (hCE-1) and carboxylesterase-2 (hCE-2). The abundant expression of hCE-1 in Caco-2 cells was supported by reverse transcription-polymerase chain reaction. In the normal human small intestine, hCE-2 is abundantly present, although the human liver expresses much higher levels of hCE-1 and lower levels of hCE-2. The expression pattern of carboxylesterases in Caco-2 cells is completely different from that in human small intestine but very similar to that in human liver. Since the substrate specificity of hCE-1 differs from that of hCE-2, it is suggested that the prediction of human intestinal absorption using Caco-2 cell monolayers should be performed carefully in the case of ester-and amide-containing drugs such as prodrugs.

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Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

Vasohibin is thought to be an important negative feedback regulator of angiogenesis that is selectively induced in endothelial cells by VEGF. Here, we assessed the role of vasohibin on HIF‐1α expression under oxidative stress induced by hydrogen peroxide (H2O2) in HUVEC. VEGF induced significant cell growth that was associated with an increase in vasohibin expression. Following H2O2‐pretreatment, VEGF further increased cell growth but this was contrastingly associated with a decrease in vasohibin expression when compared with VEGF alone. Interestingly, vasohibin inhibited cell proliferation through degradation of HIF‐1α expression during H2O2‐pretreatment. Furthermore, vasohibin elevated the expression of prolyl hydroxylase (PHD). These results suggest that vasohibin plays crucial roles as a negative feedback regulator of angiogenesis through HIF‐1α degradation via PHD.

Isoelectric-focusing analysis on an Ampholine/polyacrylamide-gel plate revealed that met-form haemoglobins are present as half-oxidized haemoglobins such as the (a2+fl3+)2 and (a3+,f2+)2 forms rather than as methaemoglobin in the erythrocytes of normal human adults and also of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase.It seems to be generally accepted that the oxidized haemoglobin, which represents less than 1 % of the total haemoglobins in normal human erythrocytes, is methaemoglobin (Bodansky, 1951). However, analytical investigations into whether the met-form haemoglobin is fully oxidized or partially oxidized have not been reported. Previously we showed that met-form haemoglobin in glucose-depleted erythrocytes is not MetHb, but half-oxidized haemoglobin (Tomoda et al., 1978a). This finding suggests that fully oxidized haemoglobin (methaemoglobin) might be absent in human erythrocytes even under physiological conditions. To study the possibility further, we carried out the identification of the met-form haemoglobins in intact human erythrocytes of normal subjects and also from a patient with hereditary methaemoglobinaemia due to deficiency of NADHcytochrome b5 reductase, by using isoelectricfocusing analysis.As a result, it was found that these contain halfoxidized haemoglobins such as the (a2+,f3+)2 and (a3+fl+)2 forms rather than MetHb as met-form haemoglobins. On the basis of the results, the mechanism of haemoglobin oxidation in human erythrocytes is discussed. ExperimentalBlood samples (5 ml) were freshly obtained without anticoagulant from 20 normal human adults (with their informed consent). Clotting ofthe blood samples was not observed, since silicone-coated injectors and glass tubes were used for collecting the blood and the Abbreviations used: HbA, haemoglobin A; MetHb, methaemoglobin. Throughout this paper the haemoglobins are, for convenience, referred to as though they were the unoxygenated forms.Vol. 181 samples were stood in an ice bath. The samples were centrifuged at 10000 rev./min (ray. 7.2 cm) for 1 min within 30 min. After removal of the plasma, the erythrocytes were suspended in 0.9 % NaCl solution and centrifuged at 10000rev./min for 1 min. The erythrocytes obtained by this procedure were haemolysed by the addition of 5 vol. of ice-cold distilled water. The haemolysates were further centrifuged at 10000rev./min for 20min to remove 'ghosts', and used for the experiments.The heparinized blood of a patient with hereditary methaemoglobinaemia due to deficiency of NADHcytochrome b5 reductase was also treated by the same procedure as stated above within 4h of drawing the sample, and used for the experiments. [The ferricyanide reductase and NADH-cytochrome b5 reductase activities in the haemolysate, which were measured by the methods of Hegesh & Avron (1967) and Sugita et al. (1971) respectively, were extremely low.]Concentrations of met-form haemoglobins were determined as described by Evelyn & Malloy (1938). The contents of met-form haemo...

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Pyriproxyfen is a juvenile hormone mimic of vital importance for insect development with little risk to humans. This study was performed to investigate whether large doses of pyriproxyfen affect the immune response in mammals. Mice were immunized thrice with ovalbumin in 5% ethanol, with or without pyriproxyfen or alum. Large doses of pyriproxyfen (9 or 15 mM) significantly enhanced specific total IgG immune response. This enhancement was no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, pyriproxyfen induced higher titers of IgG2a and enhanced tumor necrosis factor-alpha and gamma-interferon responses whereas alum induced IgG1 with enhanced interleukin-4 and -10. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that of alum.

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