Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

The locus controlling liver GM1(NeuGc) expression is mapped 1 cM centromeric to H-2K

The polymorphic variation of liver GM1 (NeuGc) ganglioside was found in inbred strains of the mouse. The genetic analysis using C57BL/10 (GM1-negative) and SWR (GM1-positive) mice revealed that a single autosomal gene (Ggm-1) was involved in the expression of liver GM1(NeuGc) and that C57BL/10 mice lacking GM1(NeuGc) expression carried a defective gene on Ggm-1. Since our previous study on H-2 congenic mice indicated that Ggm-1 was linked to the H-2 complex, in this study we measured recombination frequencies among Ggm-1, Go-1 and H-2K in the backcross progeny between (C57BL/10 × SWR)F₁ and C57BL/10. Ggm-1 was mapped 1 cM centromeric to H-2K on chromosome 17.Abbreviations used in this paper GM1(NeuGc) Gal1-3GalNAc1-4 (NeuGc2-3)Gal1-4Glc1-ceramide - GM2(NeuGc) Gal1-4(Neu Gc2-3)Gal1-4Glc1-ceramide - GM3(NeuGc) NeuGc2-3Gal1-4 Glc1-ceramide - GD1a(NeuGc) NeuGc2-3Gal1-3GalNAc1-4 (NeuGc2-3)Gal1-4Glc1-ceramide     

Monoclonal Antibodies Specific for NADH-Ferricyanide Reductase Domain of Spinach Leaf Nitrate Reductase

Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)     

Inhibition of RNA synthesis in embryo of Xenopus laevis by protease inhibitor

We have studied the role of proteases during the development of Xenopus laevis embryos with the aid of protease inhibitors. The activity of proteases was found to be only minimal in the unfertilized egg and during the initiation of development, but activity began to increase at the morula stage. When the activity of proteases was inhibited by antipain, an inhibitor of endopeptidase activity, RNA synthesis in the embryo was inhibited. To examine the relationship between the inhibitory effect of antipain on protease activity and its effect on RNA synthesis, antipain was reduced with NaBH4 to inactivate its protease inhibitory activity. The reduced antipain did not inhibit RNA synthesis in the embryo. Antipain effectively inhibited synthesis of both rRNA and poly(A)+RNA but not 4S RNA. We therefore suggest that protease activity plays an important role in the initiation and/or continuation of RNA synthesis.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Loss of androgen dependency with preservation of functional androgen receptors in androgen-dependent mouse tumor (Shionogi Carcinoma 115).

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.     

Changes of carp myosin subfragment-1 induced by temperature acclimation

Summary Heavy meromyosin subfragment-1 (S1) was prepared by -chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca²⁺- and K⁺ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 mol P_i·min^-1·mg^-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (K_D) of S1 from cold-acclimated carp was 32.1x10^-4· s^-1, compared to 13.2x10^-4·s^-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg²⁺-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s^-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - K _m apparent dissociation constant - P _i inorganic -phosphate - PMSF phenylmethane-sulfonyl fluoride - S ₁ heavy meromyosin subfragment-1 - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TPCK N-tosyl-l-phenylalanyl chloromethyl ketone - V _max maximum initial velocity     

Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 m in diameter) and less electron-dense than the D-granules (less than 0.6 m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.     

In vivo activation of cirral movement in Stylonychia by calcium

Summary Motor responses of cirri (= organelles consisting of bundles of cilia) in the protozoan Stylonychia are elicited by positive or negative shifts of the membrane voltage from its resting state. The same responses are evoked at voltages near the Ca²⁺ equilibrium potential (E_Ca) applying extremely positive steps under voltage clamp. Motor responses recorded at large positive voltages approaching E_Ca from the negative side corresponded to cirral activation following physiological depolarization from the resting potential (DCA). The hyperpolarization-induced activation of the cirri (HCA) was documented during step potentials positive to E_Ca, suggesting that the observed HCA of the cirri resulted from an efflux of Ca²⁺ from the ciliary space as compared with DCA, which is related to Ca²⁺ influx. The ciliary responses were graded functions of the rising outward or inward driving force for Ca²⁺. Slopes of reciprocal plots of response latencies near E_Ca as a function of membrane potential indicate a removal of Ca²⁺ during HCA which exceeds the free intraciliary Ca²⁺ content at rest. It is suggested that this excess Ca²⁺ is released from axonemal binding sites.     

Characteristics of progestin binder in hypertrophic human prostate

The human prostate contains a protein which binds with progesterone in a high affinity and low capacity fashion. Characteristics of the progestin-binding protein in the prostate have been disputable; whether it is progesterone receptor or not. Therefore, the characteristics of the progestin binder in the benign hypertrophic human prostate was examined in the present study. After photoaffinity labeling with 3H-R 5020, the binder in the prostate migrated to the site of 42K on polyacrylamide gel electrophoresis under denaturing conditions, and the mobility was apparently different from that of the progesterone receptor in the human uterine endometrium. There was no protein in the prostate immunoreacted with a monoclonal antibody raised against the human progesterone receptor. It was concluded that the progestin-binding protein in the human prostate was different from the progesterone receptor observed in the female human organs.