Hypertonicity-induced expression of aquaporin 3 in MDCK cells

Aquaporins (AQPs) are water channel proteins that participate in water transport. In the principal cells of the kidney collecting duct, water reabsorption is mediated by the combined action of AQP2 in the apical membrane and both AQP3 and AQP4 in the basolateral membrane, and the expression of AQP2 and AQP3 is regulated by antidiuretic hormone and water restriction. The effect of hypertonicity on AQP3 expression in Madin-Darby canine kidney (MDCK) epithelial cells was investigated by exposing the cells to hypertonic medium containing raffinose or NaCl. Northern blot and immunoblot analyses revealed that the amounts of AQP3 mRNA and AQP3 protein, respectively, were markedly increased by exposure of cells to hypertonicity. These effects were maximal at 12 and 24 h, respectively. Immunofluorescence and immunoelectron microscopy also demonstrated that the abundance of AQP3 protein was increased in cells incubated in hypertonic medium and that the protein was localized at the basolateral plasma membrane. These results indicate that the expression of AQP3 is upregulated by hypertonicity.     

Studies on the phosphorylation of protein kinase B by Ca(2+)/calmodulin-dependent protein kinases

Protein kinase B (PKB) was recently reported to be activated on the phosphorylation of Thr(308) by Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaM-kinase kinase alpha), suggesting that PKB was regulated through not only the phosphoinositide 3-kinase pathway but also the Ca(2+)/calmodulin protein kinase pathway. The activation of PKB by CaM-kinase kinase alpha was as high as 300-fold after incubation for 30 min under the phosphorylation conditions, and still increased thereafter, suggesting that the maximal activation of PKB on phosphorylation of the Thr(308) residue is several hundred fold. On the other hand, the V(max) value of CaM-kinase kinase alpha for the phosphorylation of PKB was more than two orders of magnitude lower than that for CaM-kinase IV, although the K(m) values for PKB and CaM-kinase IV were not significantly different, raising the question of whether or not PKB is a physiological substrate of CaM-kinase kinase alpha. Besides CaM-kinase kinase alpha, CaM-kinase II also remarkably activated PKB. However, the specific activities of CaM-kinase kinase alpha and CaM-kinase II as to the activation of PKB were more than three orders of magnitude lower than that of 3-phosphoinositide-dependent protein kinase 1 (PDK1).     

Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.     

Development of dendritic epidermal T cells with a skewed diversity of gamma delta TCRs in V delta 1-deficient mice

One of the most intriguing features of gammadelta T cells that reside in murine epithelia is the association of a specific Vgamma/Vdelta usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the "first wave" Vgamma5+ fetal thymocytes and overwhelmingly express the canonical Vgamma5/Vdelta1-TCRs lacking junctional diversity. Targeted disruption of the Vdelta1 gene resulted in a markedly impaired development of Vgamma5+ fetal thymocytes as precursors of DETCs; however, gammadeltaTCR+ DETCs with a typical dendritic morphology were observed in Vdelta1-/- mice and their cell densities in the epidermis were slightly lower than those in Vdelta1+/- epidermis. Moreover, the Vdelta1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vgamma5+ DETCs were predominant in the Vdelta1-/- epidermis, though Vgamma5- gammadeltaTCR+ DETCs were also detected. The Vgamma5+ DETCs showed a typical dendritic shape, gammadeltaTCR(high), and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vgamma5- gammadeltaTCR+ DETCs showed a less dendritic shape, gammadeltaTCR(low), and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vgamma/Vdelta-chain usage but requires expression of a limited diversity of gammadeltaTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.     

TCR-independent activation of extrathymically developed, self antigen-specific T cells by IL-2/IL-15

Naive intrathymically developed T cells, which express foreign Ag-specific TCR, do not express IL-2R. After antigenic stimulation, they express high affinity IL-2R, which enables IL-2 to be used as an autocrine growth factor. On the contrary, extrathymically developed T cells, which express self Ag-specific TCR but are unresponsive to antigenic stimulation, spontaneously express low affinity IL-2R. In this study, we compared the responses of these two subsets of T cells to IL-2R stimulation and examined the influences of TCR-mediated signaling on the responses. IL-2 or IL-15 augmented the proliferative response of Ag-stimulated, intrathymically developed T cells. On the other hand, extrathymically developed T cells proliferated in response to IL-2 or IL-15, independently of Ag stimulation. Furthermore, both IL-2 and IL-15 induced IFN-gamma production of these T cells, which is strikingly augmented by the presence of IL-12. These results revealed functional differences between intrathymically developed, foreign Ag-specific T cells and extrathymically developed, self Ag-specific T cells. The latter can be activated by some inflammatory cytokines, in an Ag-independent manner, similar to NK cells.     

Myocardial Depolarizing Response to Glutamate in the Myogenic Heart of the Branchiopod Crustacean Triops longicaudatus

Fine structure of the heart and the effects on the heartbeat of some transmitter candidates in crustacean cardioregulatory system were examined in the myogenic heart of the branchiopod crustacean Triops longicaudatus. Electron microscopy revealed that, in each myocardial cell, myofibrils are confined in the part facing the epicardium and intercalated disks are present between the myofibrillar regions of adjacent myocardial cells. No neural elements were found in the heart, suggesting lack of extrinsic cardioregulatory nerves from the central nervous system. Gamma aminobutyric acid and acetylcholine produced no detect-able changes in the myogenic activity of the heart at concentrations up to 10(-3) M, respectively. Glutamate induced a depolarizing membrane response in the cardiac muscle with a threshold concentration of approximately 1x10(-5) M. The amplitude of the depolarizing response was concetration-dependent and saturated at approximately 1x10(-4) M. The myogenic activity of the heart increased in frequency with glutamate of less than approximately 3x10(-5) M. With higher dose of glutamate, action potential adaptation occurred in the cardiac muscle and the heart exhibited a systolic arrest.     

Magnetic phagosome motion in J774A.1 macrophages: influence of cytoskeletal drugs

The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome.     

New EIA technique for tyrosinase in human melanocytes and its application

The expression of tyrosinase in melanocytes relates to skin pigmentation or depigmentation. Although many types of drugs with whitening effects are well known, neither the definite effect nor the mechanism underlying the effect has been elucidated. In this study, we attempted to develop the rapid and simple EIA technique for tyrosinase protein, then this technique was applied to normal human cultured melanocytes. When primary antibody and tyrosinase were incubated in non-coated 96-well microtitre plates for 48 hours at 4 degrees C, then the solution in tyrosinase-coated plate was further incubated for another 1 hour at 37 degrees C. Thus the best results were obtained. The developed EIA system could detect authentic tyrosinase until 0.1-1.0 ng/mL. This EIA technique could also be applied to human cultured melanocytes. The melanocytes cultured with endothelin-1 induced tyrosinase like immune reactive protein. The protein induction with endothelin-1 was suppressed by BQ 123, ETa receptor antagonists. The simple EIA technique developed for tyrosinase may give a clue to determination of the onset mechanisms underlying pigmental diseases of the skin as well as the mechanisms underlying the effects of various whitening drugs.     

Nuclear Localization of Brain-type Glycogen Phosphorylase in Some Gastrointestinal Carcinoma

Our previous reports have demonstrated frequent and strong expression of glycogen phosphorylase (EC 2.4.1.1) activity mainly in the cytoplasm of gastric carcinoma. Although previous studies have suggested the phosphorylase glyco-syltransferase system to be in the nucleus from enzyme histochemical analyses, intranuclear localization of the phosphorylase has not been fully established. The aims of the present study are to investigate the nuclear localization of glycogen phosphorylase and to identify the isoform of phosphorylase in the nucleus of gastrointestinal carcinoma. The activity of glycogen phosphorylase in carcinoma cells corresponding to the nucleus was demonstrated using enzyme cytochemical analysis. The phosphorylase activity coincided with localization revealed by immunocytochemistry using affinity-purified specific anti-human brain-type glycogen phosphorylase antibody. The isoform expressed in the nuclei of carcinoma cells was identified as bei ng only the brain type according to a polymerase chain reaction-based assay using RNA obtained from gastric carcinoma cells and primers specific to muscle, liver and brain types of glycogen phosphorylase. The intranuclear localization of the brain-type isoform was confirmed by immunoelectron microscopical analyses. Further investigation to examine the nuclear localization in human carcinoma tissue (145 and 25 specimens with gastric and colonic carcinoma respectively) was carried out by immunohistochemistry using specific anti-brain-type antibody. Nuclear immunostaining was observed in seven cases out of 145 gastric carcinoma. The present study is the first to clarify the nuclear localization of glycogen phosphorylase with enzymatic activity in gastrointestinal carcinoma. The isoform of the enzyme expressed in the carcinoma was identified as the brain type. These results warrant further studies on the mechanisms for transporting the large molecule of brain-type glycogen phosphorylase to nuclei and its function in the nucleus of carcinoma cells.     

Isolation of a Periplasmic Molecular Chaperone-Like Protein of Rhodobacter sphaeroides f. sp. denitrificans That Is Homologous to the Dipeptide Transport Protein DppA of Escherichia coli

A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph Rhodobacter sphaeroides f. sp. denitrificans, in a manner similar to that of the Escherichia coli chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333–339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of E. coli. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.