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Key points The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3- concentrations as high as 140 mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts. The model was readily able to secrete 140 mm HCO3- . Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3- concentrations is to minimize the secretion of Cl- ions. These findings help to clarify the mechanism responsible for pancreatic HCO3- secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. Abstract A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3- ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+ -HCO3- cotransporter (NBC1) and apical Cl- /HCO3- exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl- and HCO3- permeabilities (CFTR), and reducing the activity of the basolateral Cl- /HCO3- exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 mm HCO3- at a rate of ∼3 nl min-1 mm-2 , which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl- /HCO3- exchange via SLC26A6 at the apical membrane were able to support a HCO3- -rich secretion. Raising the HCO3- /Cl- permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3- concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl- concentration following cAMP stimulation and thereby maximizing the secreted HCO3- concentration. The addition of a basolateral Na+ -K+ -2Cl- cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl- and resulted in a lower secreted HCO3- concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl- secretion is the main requirement for secreting 140 mm HCO3- .

Aim Glycosylation changes induce various types of biological phenomena in human diseases. N-Acetylglucosaminyltransferase V (GnT-V) is one of the most important glycosyltransferases involved in cancer biology. Recently, many researchers have challenged studies of lipid metabolism in cancer. To elucidate the relationships between cancer and lipid metabolism more precisely, we investigated the effects of GnT-V on lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on hepatic triglyceride production. Methods We compared lipid metabolism in GnT-V transgenic (Tg) mice with that of wild-type (WT) mice fed with normal chow or a choline-deficient amino acid-defined (CDAA) diet in vivo. HepG2 cells and GnT-V transfectants of Hep3B cells were used in an in vitro study. Results Serum triglyceride levels and hepatic very low-density lipoprotein (VLDL) secretion in Tg mice were significantly elevated compared with that of WT mice. Hepatic lipogenic genes (Lxrα, Srebp1, Fas and Acc) and VLDL secretion-related gene (Mttp1) were significantly higher in Tg mice. Expression of these genes was also significantly higher in GnT-V transfectants than in mock cells. Knockdown of GnT-V decreased, while both epidermal growth factor and transforming growth factor-β1 stimulation increased LXRα gene expression in HepG2 cells. Finally, we found that the blockade of VLDL secretion by CDAA diet induced massive hepatic steatosis in Tg mice. Conclusion Our study demonstrates that enhancement of hepatic GnT-V activity accelerates triglyceride synthesis and VLDL secretion. Glycosylation modification by GnT-V regulation could be a novel target for a therapeutic approach to lipid metabolism.

VLCAD deficiency is an autosomal recessive disorder caused by a defect of fatty acid oxidation. The phenotype is classified into three clinical forms on the basis of the onset of symptoms: a severe form with neonatal onset; a milder form with childhood onset; and a late-onset form. The neonatal form is the most common, and has a higher mortality rate than the others. We report the case of a newborn infant with VLCAD deficiency who developed ventricular fibrillation, which was successfully treated by intensive care, but who suddenly died after a respiratory syncytial virus infection. Early institution of i.v. glucose treatment and active immunization with vaccine, such as palivizumab (anti-RSV mAb), may be important to reduce the frequency and severity of life-threatening episodes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pleiotropic functions. We report here that PACAP-deficient (PACAP-/-) mice showed increased immobility in a forced swimming test, which was reduced by the antidepressant desipramine, to a similar extent as in wild-type mice. The atypical antipsychotic risperidone and the selective serotonin (5-HT)(2) antagonist ritanserin normalized the depression-like behavior in PACAP-/- mice. The 5-HT(2) agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine-induced 5-HT syndrome was exaggerated in PACAP-/- mice, which suggests a 5-HT(2)-receptor-dependent mechanism in the depression-like behavior. The circadian rhythm of plasma corticosterone and body core temperature was significantly flattened in the mutants. mRNA expression of glucocorticoid receptor was reduced in the mutant hippocampus. The present results suggest that alterations in PACAP signaling might contribute to the pathogenesis of certain depressive conditions amenable to atypical antipsychotic drugs.

LEAFY COTYLEDON 1 (LEC1) plays vital roles in the regulation of seed maturation in Arabidopsis. LEC1 encodes a homolog of yeast HAP3 or mammalian NF-YB/CBF-A subunit of trimeric CCAAT binding factor (CBF). Among the nine paralogs of NF-YB in Arabidopsis, LEC1-LIKE (L1L) is most closely related to LEC1, and can complement the lec1 mutation when expressed under the control of the LEC1 promoter. Although the nature of the B3-type seed maturation regulators as transcription factors have been investigated, knowledge of the molecular action of LEC1 is limited. When co-expressed with NF-YC2 in the presence of ABA, we found that LEC1 or L1L, but not other NF-YBs, activated the promoter of CRUCIFERIN C (CRC), which encodes a seed storage protein. However, additional expression of an NF-YA subunit interfered with the activation. The LEC1/L1L-[NF-YC2] activation depended on ABA-response elements present in the promoter, which led to the finding that LEC1/L1L-[NF-YC2] can strongly activate the CRC promoter in the absence of ABA when co-expressed with a seed-specific ABA-response element (ABRE)-binding factor, bZIP67. Functional coupling of LEC1/L1L-[AtNF-YC2] and bZIP67 was also observed in the regulation of sucrose synthase 2 (SUS2). Immunoprecipitation experiments revealed that L1L and bZIP67 formed a protein complex in vivo. These results demonstrate a novel plant-specific mechanism for NF-Y subunit function that enables LEC1 and L1L to regulate a defined developmental network.

The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.

Aim: We evaluated the usefulness of Doppler ultrasonography (DUS) for the analysis of tumor hemodynamics in small hepatocellular carcinoma (HCC). Methods: We compared Doppler ultrasound (DUS) findings with angiography-assisted computed tomography (Angio-CT) such as CT during arterial portography and during hepatic arteriography in the evaluation of the intratumoral hemodynamics, and with pathologic findings in 45 small HCC nodules (≤3.0 cm in diameter) of 43 patients. DUS flow pattern of each nodule was categorized into three types: afferent continuous flow (Type 1), afferent pulsatile flow with afferent continuous flow (Type 2), and afferent pulsatile flow without afferent continuous flow (Type 3). Intratumoral blood supply was determined by Angio-CT, and pathologic findings were evaluated on resected or biopsied specimen. Results: Based on Angio-CT findings, Type 1 nodules showed decreased arterial blood supply (ABS) without decreased portal blood supply (PBS). Type 2 nodules showed unchanged ABS but decreased PBS. Type 3 nodules showed both increased ABS and decreased PBS. DUS findings well represented blood supply of HCC evaluated by Angio-CT. In addition, all Type 1 and 2 nodules were well-differentiated HCC, and all Type 3 nodules were moderately or poorly differentiated HCC; DUS findings well reflected differentiation of HCC. Conclusions: DUS is a non-invasive imaging method and can be used for the evaluation of the stage of malignancy of small HCC.

Ethanol is the leading cause of pancreatitis; however, its cellular effects are poorly understood. We examined the direct effects of ethanol in the concentration range 0.1–30 mM, i.e. relevant to usual levels of drinking, on fluid secretion from guinea-pig pancreatic duct cells. Fluid secretion was continuously measured by monitoring the luminal volume of interlobular duct segments isolated from the guinea-pig pancreas. [Ca2+]i was estimated by microfluorometry in duct cells loaded with fura-2. Ethanol at 0.3–30 mM significantly augmented fluid secretion stimulated by physiological (1 pM) or pharmacological (1 nM) concentrations of secretin. It augmented dibutyryl cAMP-stimulated fluid secretion but failed to affect spontaneous or acethylcholine-stimulated secretion. Ethanol at 1 mM shifted the secretin concentration-fluid secretion response curve upwards and raised the maximal secretory response significantly by 41 %. In secretin-stimulated ducts, 1 mM ethanol induced a transient increase in [Ca2+]i that was dependent on the presence of extracellular Ca2+. Ethanol failed to augment secretin-stimulated secretion from ducts pretreated with an intracellular Ca2+ buffer (BAPTA) or a protein kinase A inhibitor (H89). In conclusion, low concentrations of ethanol directly augment pancreatic ductal fluid secretion stimulated by physiological and pharmacological concentrations of secretin, and this appears to be mediated by the activation of both the intracellular cAMP pathway and Ca2+ mobilization.

Metallic biomaterials implanted in a human body may corrode and wear, releasing metal ions and debris which may induce adverse reactions such as inflammation, allergy, neoplastic formation, developmental malformation, etc. Mutagenicity is a very fundamental and important toxicity related to carcinogenicity and reproductive/developmental toxicity because the damages to genes or DNA can be a cause of carcinogenesis and developmental abnormalities. However, available mutagenic data on metallic ions and compounds are restricted to the number of elements. Therefore, to obtain the systematic data necessary for metal ion mutagenicity, 41 metal salts encompassing 36 metals and 5 metallic elements tested with different valences, were evaluated on their mutagenicity by a microbial test, the umu test. As a result, K2Cr2O7, RhCl3, IrCl4, and MgCl2 are positive without metabolic activation. Concentrations having the maximum mutagenic effect (Cmax) are 9.65 × 10−5, 1.00 × 10−4, 3.11 × 10−3, 4.12 × 10−3 mol · L−1, respectively. CuCl2, VCl3, CuCl, RhCl3, K2Cr2O7, and IrCl4 are positive with metabolic activation by S-9 mix with Cmax of 1.60 × 10−5, 3.91 × 10−5, 1.57 × 10−4, 2.00 × 10−4, 3.86 × 10−4, 1.56 × 10−2 mol · L−1, respectively. Thirty-five metal salts were negative for tests performed both with and without metabolic activation, whereas it was impossible to evaluate the mutagenicity of MoCl5 and ZrCl4 by the umu test because of their colorimetric reaction to testing reagents. © 2001 Wiley Periodicals, Inc. J Biomed Mater Res 59: 176–183, 2002

Permeabilities of the luminal and basolateral membranes of pancreatic duct cells to CO2 and HCO3− were examined in interlobular duct segments isolated from guinea-pig pancreas. Intracellular pH (pHi) was measured by microfluorometry in unstimulated, microperfused ducts loaded with the pH-sensitive fluoroprobe 2′7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). When HCO3−/CO2 was admitted to the bath, pHi decreased transiently as a result of CO2 diffusion and then increased to a higher value as a result of HCO3− uptake across the basolateral membrane by Na+-HCO3− cotransport. When HCO3−/CO2 was admitted to the lumen, pHi again decreased but no subsequent increase was observed, indicating that the luminal membrane was permeable to CO2 but did not allow HCO3− entry to the cells from the lumen. Only when the luminal HCO3− concentration was raised above 125 mm was HCO3− entry detected. The same was true of duct cells stimulated with forskolin. Recovery of pHi from an acid load, induced by exposure to an NH4+ pulse, was dependent on basolateral but not luminal Na+ and could be blocked by basolateral application of methylisobutylamiloride and H2DIDS. This indicates that the Na+-H+ exchangers and Na+-HCO3− cotransporters are located exclusively at the basolateral membrane. In the presence of HCO3−/CO2, substitution of basolateral Cl− with glucuronate caused larger increases in pHi than substitution of luminal Cl−. This suggests that the anion exchanger activity in the basolateral membrane is greater than that in the luminal membrane. We conclude that the luminal and basolateral membranes are both freely permeable to CO2, but while the basolateral membrane has both uptake and efflux pathways for HCO3−, the luminal membrane presents a significant barrier to the re-entry of secreted HCO3−, largely through the inhibition of the luminal anion exchanger by high luminal HCO3− concentrations. The ductal system of the exocrine pancreas produces a bicarbonate-rich fluid secretion in response to secretin and some other stimuli. However, there are significant species-dependent variations in the pattern of pancreatic HCO3− secretion in vivo. In the guinea-pig (Padfield et al. 1989) and in several other species, including dog, cat and human (Case & Argent, 1993), the HCO3− concentration of the juice may reach 140–150 mm during maximal stimulation with secretin. In the rat, however, the highest HCO3− concentration is about 70 mm (Sewell & Young, 1975). We have recently reported that interlobular duct segments isolated from guinea-pig pancreas secrete a HCO3−-rich fluid (> 130 mm) during stimulation with 10 nm secretin (Ishiguro et al. 1998), which indicates that the in vivo behaviour of pancreatic ducts is well preserved in this in vitro preparation. In order to maintain such a high concentration of HCO3− in pancreatic juice during sustained secretion, pancreatic duct cells must possess the following characteristics. (1) HCO3− must be actively accumulated in the cell by transport across the basolateral membrane. We have previously shown that Na+-HCO3− cotransport plays a dominant role in this process (Ishiguro et al. 1996a). (2) The luminal membrane must possess a mechanism which specifically transports HCO3− into the lumen. In a previous paper (Ishiguro et al. 1996b), we demonstrated a secretin-stimulated HCO3− efflux pathway in the luminal membrane that has yet to be identified. (3) The luminal membrane must not contain transporters that would allow HCO3− secreted into the lumen to re-enter the cell. To compare the passive permeability characteristics of the basolateral and luminal membranes of guinea-pig pancreatic duct cells to extracellular CO2 and HCO3−, we have examined the changes in intracellular pH that occur in unstimulated, microperfused, interlobular duct segments when either the basolateral or the luminal membrane is exposed to changes in HCO3− or CO2 concentration.

Quantitative evaluation of a material's affinity for cells is essential to understanding cell-material interaction inside a body and it is also necessary for the development of new biomaterials with superior biocompatibility. In the present study, the shear force and the total energy necessary to detach a single murine fibroblast L929 adhering to glass, polystyrene, and fibronectin- or collagen-coated polystyrene were measured directly by applying a lateral force, using a cantilever, to the cell. The projected area of the cell was also measured, and then cell adhesive shear strength and cell detachment surface energy were determined by dividing the shear force and the total energy by the area. Among these four materials, the cells on collagen-coated polystyrene have the highest cell adhesive shear strength and cell detachment surface energy (1500 Pa and 29 pJ on average, respectively), followed by the cells on fibronectin-coated polystyrene (1000 Pa and 16 pJ, respectively). The cells on glass and polystyrene had almost the same cell adhesive shear strength and cell detachment surface energy (420-670 Pa and 7-11 pJ, respectively). These observations suggest that cell adhesive shear strength and cell detachment surface energy depend on the number of the bindings between the cell and a material's surface rather than on the strength of each binding.

The release of metal ions due to active oxygen species generated by macrophages (Mphi) phagocytosing high-density polyethylene (HDPE) particles was studied in vitro to investigate the mechanism behind the release of metal ions from titanium implants into nearby tissues in the absence of wear and fretting in vivo. To determine the effects of Mphis on metal ion release, titanium disks were immersed in different solutions and the titanium ions released from the titanium disks into each solution were quantified. The results revealed that active oxygen species generated by Mphis induced the metal ion release. In particular, the ion release was accelerated with HDPE because the Mphis that phagocytosed HDPE generated more active oxygen species than Mphis that did not phagocytose any HDPE. Metal ions were also released by organic species in the absence of Mphis. These are some of the causes for metal ion release from titanium implants in the absence of wear and fretting in vivo.

Systematic cytotoxicity evaluation of various metallic elements may contribute to the development of new metallic biomaterials with superior biocompatibility. It is generally reported that the cytotoxicity of a chemical differs with cell lines. However, our previous study revealed a high correlation in the cytotoxicity of 43 metal salts between two murine cell lines. If there is any generic tendency toward metal salt cytotoxicity for many kinds of cells, that information is helpful for the determination of the chemical composition of new metallic biomaterials that are expected to have lower cytotoxicity. In this study, the cytotoxicity of 12 metal salts was evaluated using four cell lines, and the results were compared, including those for two other cell lines obtained in our previous study. A metal salt concentration that reduced cell viability to 50% of that without any metal salt (IC(50)) was used as an index to compare the metal salt cytotoxicity between cell lines. The correlation was statistically proved by the IC(50)s of 12 metal salts among these cell lines (p < 0.01), suggesting the existence of a generic tendency to metal salt cytotoxicity beyond cell lines. The metal salt order of toxicity from the highest was K(2)Cr(2)O(7), AgNO(3), VCl(3), SbCl(3), CuCl(2), CoCl(2), NiCl(2), ZnCl(2), Cr(NO(3))(3), FeCl(3), TiCl(4), and Al(NO(3))(3). The sensitivity for metal salt cytotoxicity differed with cell lines; IMR-32 had the highest sensitivity among the six cell lines.

Metallic biomaterials are generally used for replacement of structural components of the human body such as bones, joints, and tooth roots. When they are implanted inside a body, metallic biomaterials may corrode and/or wear, releasing metal ions and debris which may have toxic effects on tissues and organs. Since it is important for biomaterials to have no toxicity against a living body, a systematic and quantitative evaluation of the cytotoxicity of metallic elements is required for the development of new metallic biomaterials with superior biocompatibility. In this study, the cytotoxicity of 43 metal salts were evaluated by the colony formation method using two kinds of cultured cells. The effects of the difference in valence numbers of metallic elements in the salts on cytotoxicity were examined. The cytotoxicity of the salts of metallic elements' oxo acids was also investigated. As a result, the intensity of metal salts' cytotoxicity tends to be quite similar between MC3T3-E1 and L929 (the correlation coefficient of metal salts' IC50s is 0.82). The intensity of metal salts' cytotoxicity depends on the kinds of metallic elements, their chemical states, and concentrations. The IC50 of the highest toxic salt is 1.36 x 10(-6) mol L-1, which differs four orders of magnitude from the IC50 of the lowest toxic salt. K2Cr2O7, CdCl2, VCl3, AgNO3, HgCl2, SbCl3, BeSO4, and InCl3 are high toxic salts in which IC50s are smaller then 10(-5) mol L-1 for both or either of the cell lines. HgCl, Tl(NO3)3, GaCl3, CuCl2, MnCl2, CoCl2, ZnCl2, NiCl2, SnCl2, IrCl4, TlNO3, CuCl, RhCl3, Pb(NO3)2, Cr(NO3)3 and Bi(NO3)3 are relatively high toxic salts in which IC50s are smaller than 10(-4) mol L-1 for both or either cell lines.

A low-molecular-mass chromium-binding substance (LMCr), which is recognized as a detoxification ligand of chromium, was isolated from the livers of rabbits injected intravenously with K2Cr2O7 (200 μmol Cr/kg body wt) as a biologically active form. LMCr appears as an anionic, organic Cr compound with a relative molecular mass of 1500. It is composed of glutamic acid or glutamine, glycine, cysteine and aspartic acid or asparagine with a Cr/amino-terminal residue ratio of 4:1. The purified LMCr (10–300 ng Cr/ml) shows in vitro activities comparable to those of glucose tolerance factor in relation to insulin action. In the presence of insulin it enhances [U-14C]glucose conversion to 14CO (23–30% up) in rat epididymal adipocytes above the value obtained with insulin alone. LMCr also stimulates the rate of [3-3H]glucose incorporation into lipid by 30–40% with insulin or by 15–23% without insulin, as compared with the basic value obtained with insulin alone or without insulin. These findings suggest that LMCr plays essential roles in both glucose metabolism and detoxification of invaded Cr in the body.