A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

Additive stimulation of hepatic putrescine production by glucagon and insulin after partial hepatectomy in rats

When rats received glucagon or insulin every 2 h after partial hepatectomy (Hx), hepatic putrescine content was increased above control levels at 6 and 12 h, respectively. When the two hormones were combined, the increased levels were additive. Hepatic ornithine decarboxylase activity was above control levels at 12 h after insulin treatment. Hepatic spermidine N1-acetyltransferase activity was enhanced at 6 h only when glucagon was dosed. Putrescine administration from 0 to 4 h or from 6 to 10 h increased hepatic DNA synthesis to similar levels 22 h after Hx. These results suggest that glucagon and insulin additively stimulate hepatic putrescine production after Hx. This may explain the cooperative stimulation of liver regeneration by both hormones.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Analysis of Flow Phenomena in Gastric Contents Induced by Human Gastric Peristalsis Using CFD

This paper uses computational fluid dynamics to simulate and analyze intragastric fluid motions induced by human peristalsis. We created a two-dimensional computational domain of the distal stomach where peristalsis occurs. The motion of the gastric walls induced by an antral contraction wave (ACW) on the wall of the computational domain was well simulated using a function defined in this study. Retropulsive flow caused by ACW was observed near the occluded region, reaching its highest velocity of approximately 12 mm/s in the narrowest region. The viscosity of the model gastric contents applied in this study hardly affected the highest velocity, but greatly affected the velocity profile in the computational domain. The shear rate due to gastric fluid motion was calculated using the numerical output data. The shear rate reached relatively high values of approximately 20 s⁻¹ in the most occluded region. The shear rate profile was almost independent of the fluid viscosity. We also simulated mass transfer of a gastric digestive enzyme (pepsin) in model gastric content when peristalsis occurs on the gastric walls. The visualized simulation results suggest that gastric peristalsis is capable of efficiently mixing pepsin secreted from the gastric walls with an intragastric fluid.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Quantitative detection of bisphenol A and bisphenol A diglycidyl ether metabolites in human plasma by liquid chromatography–electrospray mass spectrometry

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.     

Recent Advances in the use of Immobilized Lipases Directed Toward the Asymmetric Syntheses of Complex Molecules

Water-insoluble compounds can be substrates for enzymatic reactions when lipases are immobilized properly and suitable organic solvents are used. In this review, three type of lipase immobilization method and their application to the asymmetric syntheses of complex molecules are described. Lipases immobilized with Celite or synthetic prepolymers such as urethane prepolymer and photo-crosslinkable resin prepolymer have been applied for the kinetic resolution of many kinds of water-insoluble substrate.

Phospholipid-lipase aggregates with ether linkages are novel and have been found to function effectively as immobilized lipases in asymmetric hydrolysis or esterification reactions in water-saturated organic solvent. The phospholipid-lipase aggregates are considered to have a stacked bilayer based on X-ray diffraction analysis structure of the lipid in the crystalline phase.     

GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.     

Quantitative analysis of development of mitochondrial ultrastructure in differentiating mouse hepatocytes during postnatal period

Between birth and 10 days of age, the volume density (volume/unit cytoplasmic volume) of the matrix, and the surface density (area/unit cytoplasmic volume) of the inner membrane and cristae increased in both periportal and perihepatic hepatocytes, and did not differ significantly between the cells of the two zones. After 10 days of age, however, the volume density of the matrix decreased in perihepatic cells and remained unchanged in periportal cells, and, therefore, it became greater in periportal cells than in perihepatic cells in 20-day-old and adult animals. The surface density of the inner membrane and cristae decreased in the cells of both zones. Further, the hepatocyte volume increased markedly, especially in perihepatic zones between 20 days of age and the adult. The results show that, in postnatally differentiating hepatocytes, mitochondria are likely to develop during early postnatal period, then the structural heterogeneity of mitochondria arises, and hepatocyte volume increases markedly during late postnatal period after weaning. Thus, the process of postnatal hepatocyte differentiation includes such several phases of development.