Immunoelectron microscopic localization of epidermal growth factor in the eccrine and apocrine sweat glands.

We studied the localization of the epidermal growth factor (EGF) in eccrine and apocrine sweat glands with light microscopic and electron microscopic immunohistochemistry. Anti-human EGF (anti-hEGF) polyclonal antiserum and anti-hEGF monoclonal antibody (MAb) were used for the study. Light microscopic immunohistochemistry with monoclonal and polyclonal antibodies showed that hEGF-like immunoreactivity was strongly positive in the myoepithelial cells and weakly positive in the secretory cells of eccrine sweat glands. In apocrine sweat glands, it was strongly positive in the secretory cells as well as in the myoepithelial cells. Immunoelectron microscopy with polyclonal antibody showed that hEGF-like immunoreactivity was present in secretory granules of apocrine secretory cells. These granules had mitochondrion-like internal structure. No reactivity was observed on the eccrine secretory cells by immunoelectron microscopy. Neither dark cell granules nor mitochondria in eccrine secretory cells were labeled with anti-hEGF antibody. In both eccrine and apocrine sweat glands, hEGF-like immunoreactivity was diffusely present in the cytoplasm of myoepithelial cells. However, nuclei and mitochondria of myoepithelial cells were devoid of immunoreactivity for hEGF. Our observations indicate that apocrine sweat glands may secrete more hEGF in the sweat than eccrine sweat glands.     

Both cyclooxygenase and lipoxygenase inhibitor partially restore the anorexia by interleukin-1 beta.

Since the peripheral prostaglandin synthetizing system may at least partly involved in the anorexia that follows central interleukin-1 beta (IL-1) administration, this study was undertaken to investigate the effect of ibuprofen (ip), selective cyclooxygenase blocker and AA 861, selective lipoxygenase inhibitor, on changes of food and water intake by a single injection of IL-1 (2 micrograms/rat, ip). We demonstrated that food and water intake were suppressed by peripheral administration of IL-1. Throughout the entire observation periods, suppressed food intake was partially restored to control levels by ibuprofen, while water intake completely restored. In addition, no significant differences about water/food intake were observed in the IL-1 + ibuprofen-treated groups, respectively. In the next experiment, IL-1 induced anorexia was also partially restored to the control level following pretreatment with AA 861. These results may suggest that other mechanism including lipoxygenase blocker besides prostaglandin production may be involved in IL-1 induced anorexia.     

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.     

Possible role of sodium-hydrogen antiport in acetylcholine-induced relaxation of rat aorta.

The decreased extracellular Na concentration (25mM) attenuated vasodilator effect of acetylcholine (ACh) in norepinephrine-treated aortic ring. This attenuation was greater in the low Na medium substituted by Li, which can exchange intracellular H through Na-H antiport, as compared with that substituted by choline, which cannot. 10 microM amiloride canceled the difference between the two low Na mediums. Thus the inhibition of Na-H antiport may counteract the suppressive effect of decreased intracellular Na on ACh vasodilation, suggesting a possible role of Na-H antiport in a release of vasoactive substance(s) from endothelial cells.     

A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities

Monoclonal antibody YC10 showed specificity for the phosphorylated form of human, bovine and porcine glial fibrillary acidic proteins (GFAPs) and negligible reactivity towards the dephosphorylated form of the GFAPs. Analysis of species specificity and of the epitope, determined using synthetic phosphopeptides, indicated that this antibody recognized the local phosphorylation-site sequence Thr-phosphoSer-Ala-Ala-Arg-Arg (residues 7-12 of GFAP). Making use of this antibody we developed a non-radioactive method to measure protein kinase activities. After incubation of a protein kinase with non-radioactive ATP in ninety-six wells coated with the synthetic peptide Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser-Cys (residues 3-13 of GFAP), the phosphorylated product was detected by using this mouse antibody and peroxidase-labeled goat anti-mouse IgG. This method proved to be equally as sensitive as the radioactive method for the measurement of protein kinase activities and was less affected by concentrations of ATP present in the reaction mixture.     

Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.     

Mechanism in 1,25(OH)2D3-induced suppression of helper/suppressor function of CD4/CD8 cells to immunoglobulin production in B cells

On the basis of previous data that 1,25(OH)2D3 suppressed both helper and suppressor activities of CD4 and CD8 cells in the pokeweek mitogen-stimulated culture, we examined the further effect of 1,25(OH)2D3 on both cells to define how 1,25(OH)2D3 is involved in the deterioration of their functions. 1,25(OH)2D3 suppressed the pokeweed mitogen and phytohemagglutinin-induced DNA synthesis of CD4 and CD8 cells. The suppression by 1,25(OH)2D3 of DNA synthesis was caused by a time lag in reaching maximal response. 1,25(OH)2D3 also suppressed interleukin-2 production of CD4 and CD8 cells. 1,25(OH)2D3 did not, however, affect their interleukin-2 receptor expression detected within 24 hr after phytohemagglutinin stimulation. In addition, 1,25(OH)2D3 failed to suppress DNA synthesis of CD4 and CD8 cells when cultured with a large amount of interleukin-2. Suppression by 1,25(OH)2D3 of proliferation and interleukin-2 production in CD4 and CD8 cells would bring about the decrease of their helper or suppressor functions by inhibiting their expansion or maturation.     

A new site-specific endonuclease StuI from Streptomyces tubercidicus

A new sequence-specific endonuclease, StuI, produced by Streptomyces tubercidicus KCC S-0054, was identified and partially purified. StuI recognizes the hexanucleotide "palindromic" sequence (Formula: see text), and cleaves it at the middle, producing blunt ends.