Elevation of intracellular cAMP levels by dominant active heterotrimeric G protein alpha subunits ScGP-A and ScGP-C in homobasidiomycete, Schizophyllum commune

In many fungi, the heterotrimeric G protein alpha subunits, and/or small G protein (RAS) control intracellular cAMP levels. But it is not clear which types of G proteins modulate cAMP levels in homobasidiomycete (mushrooms). To explain the mechanism, we expressed dominant active RAS (a homolog of S. cerevisiae RAS1) in homobasidiomycete Schizophyllum commune and compared the cAMP levels in the transformed clones with those of clones expressing dominant active heterotrimeric G protein alpha subunits ScGP-A, B, and C. The results demonstrated that the dominant active ScGP-A and C elevated the intracellular cAMP levels. In contrast, the dominant active S. commune RAS gene did not affect the cAMP levels, even though colony growth and formation of fruiting bodies were apparently repressed. These data suggest that the heterotrimeric G protein alpha subunits are involved in the mechanism of cAMP regulation, and that RAS modulates another signal-transduction pathway regulating cell growth and differentiation.     

Identification of peptidyl mimics of bioactive gibberellin recognized by an antibody

We screened a phage display peptide library for peptidyl mimotopes of gibberellin against anti-bioactive gibberellin antibody. The peptides obtained were grouped into two homologous sequences and their binding to the antibody was put in competition with free GA(4) but not with GA(4) methylester, suggesting that the peptides behave as mimics of GA(4). As an application, the phage display peptide was shown to work as a tracer for enzyme-linked immunosorbent assay (ELISA) analysis of GA(4).     

Decreased mRNA expression of the PTH/PTHrP receptor and type II sodium-dependent phosphate transporter in the kidney of rats fed a high phosphorus diet accompanied with a decrease in serum calcium concentration

This study investigates the phosphorus (P) homeostasis in the process of an altered parathyroid hormone (PTH) action in the kidney of rats fed a high P diet. Four-week-old male Wistar strain rats were fed diets containing five different P levels (0.3, 0.6, 0.9, 1.2 and 1.5%) for 21 days. The serum PTH concentration and urinary excretion of P were elevated with increasing dietary P level. Compared to rats fed the 0.3% P diet, the serum calcium (Ca) concentration remained unchanged, while the serum 1,25(OH)(2)D(3) concentration and urinary excretion of cAMP were elevated with increasing dietary P level in rats fed the high P diets containing 0.6-0.9% P. On the other hand, a lower serum Ca concentration was observed in rats fed the high P diets containing 1.2% or greater P. The serum 1,25(OH)(2)D(3) concentration remained unchanged in rats fed the high P diets containing 1.2% or greater P, comparison with rats fed the 0.3% P diet. The urinary excretion of cAMP and PTH/PTH-related peptide (PTHrP) receptor and type II sodium-dependent phosphate transporter (NaPi-2) mRNA in the kidney were both decreased in rats fed the high P diets containing 1.2% or greater P. In conclusion, a high P diet with subsequent decrease in serum Ca concentration suppressed the PTH action in the kidney due to PTH/PTHrP receptor mRNA down-regulation. Furthermore, an increase in the urinary excretion of P might have been caused by decreased NaPi-2 mRNA expression without the effects of PTH and 1,25(OH)(2)D(3).     

Maillard reaction rate in various glassy matrices

The Maillard Reaction (MR) rate below the glass transition temperature (T(g)) for various model glassy food systems was studied at temperatures between 40 degrees C and 70 degrees C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T(g) was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD(280)), and the MR rate (k(280)) was determined as a pseudo zero order reaction rate from the time course of OD(280). Although k(280) was described by the Arrhenius plot, the temperature dependence of k(280) was almost the same and the intercept was different among the matrices. From the comparison of k(280), it was suggested that the MR rate in glassy matrix was affected not only by the T(g), but also by the hydrogen bonding between MR reactants and glassy matrix.     

Alkyl- and alkenylresorcinols of wheat grains and their chemotaxonomic significance

Resorcinolic lipid contents and homologue compositions in extracts isolated from soft winter, soft spring and hard (durum) wheat grains were evaluated by both instrumental and chromatography means. Resorcinol concentrations determined in wheat were diverse and varied in samples harvested within two consecutive vegetative years, whereas their homologue profiles were found to be rather invariable. The predominant alkylresorcinols identified in wheat grains were saturated 1,3-dihydroxy-5-n-heneicosylbenzene and 1,3-dihydroxy-5-n-nonadecylbenzene. 1,3-Dihydroxy-5-n-heptadecylbenzene and 1,3-dihydroxy-5-n-tricosylbenzene were also determined, whereas 1,3-dihydroxy-5-n-pentadecylbenzene and 1,3-dihydroxy-5-n-pentacosylbenzene were present in these extracts only in spurious amounts. Furthermore, our results show that alk(en)ylresorcinols may be useful as chemotaxonomic markers for a distinction between soft and hard wheat plants. Cluster analysis with Ward's amalgamation algorithm and five different distance linkage types clearly discriminated particular wheats into species- and cultivar-specific clusters, whereas the use of principal component analysis allowed us to specify, which of the variables analysed were decisive. This approach may be useful for both plant breeders and taxonomists to classify wheat species/cultivars.     

Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles

-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin.     

The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.     

Effects of gender on kidney function in magnesium-deficient rats

This study investigated the gender differences in the kidney function of magnesium (Mg)-deficient rats. Male and female rats were fed a control diet or a Mg-deficient diet for 21 d. Mg-deficient diet had no significant effect on kidney calcium (Ca) or phosphorus (P) concentration in male rats, while Ca and P concentrations in female rats were significantly higher in Mg-deficient rats than in the control rats. With regard to indicators of kidney function, no significant differences in creatinine clearance and serum urea nitrogen concentration were observed among the groups. Serum albumin concentrations were significantly lower in rats fed the Mg-deficient diet than in rats fed the control diet. In both sexes, urinary albumin excretion was significantly higher in rats fed the Mg-deficient diet than in rats fed the control diet. Gender differences had no significant influence on creatinine clearance, serum urea nitrogen concentration, serum albumin concentration and urinary albumin excretion. These results suggest that gender differences have no effect on kidney function in Mg-deficient rats under the condition used.     

Human bronchial smooth muscle cell proliferation via thromboxane A2 receptor

Thromboxane A2 receptor (TP) mediates bronchial smooth muscle cell (BSMC) contraction, airway hyperresponsiveness, and airway inflammation in patients with asthma. In the present study, a pathogenic role of TP activation in airway remodeling was examined using primary cultures of human BSMC. A TP agonist, I-BOP, concentration-dependently enhanced not only bromodeoxyuridine (BrdU) uptake but also cell proliferation of BSMC. A TP-selective antagonist, AA-2414, blocked the effects of I-BOP on both BrdU uptake and cell proliferation. I-BOP-induced BrdU uptake was significantly blocked by two non-selective tyrosine kinase inhibitors, genistein and herbimycin A, or a Src family tyrosine kinase inhibitor, PP2, but not by an inhibitor of epidermal growth factor (EGF) receptor-associated tyrosine kinase, AG1478. In conclusion, TP receptor activation causes DNA synthesis and cell proliferation of human BSMC by activating tyrosine kinases including Src, but not by EGF receptor transactivation.