X-ray microanalysis was used to investigate whether cAMP- and/or Ca²⁺-activated regulation of chloride and potassium efflux is expressed in primary cultures of sweat gland duct cells. The effects of extracellular UTP and ATP on the duct cells, and the signalling system involved in the response to ATP was also studied. Primary cultures from duct cells of human sweat glands responded to 1 μM carbachol, 2 μM of the Ca²⁺ ionophore A23187, or 5 mM 8-bromo-cAMP stimulation for 5 min, resulting in a decrease in cellular Cl and K concentrations. 50 μM 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), a Cl" channel blocker, can inhibit the decrease in Cl concentration induced by cAMP. Extracellular (200 μM) ATP caused a decrease of Cl and K in cultured duct cells, while (200 μM and 2 mM) UTP was ineffective. Both the phosphoinositidase C inhibitor TJ73122 (10 μM) and the absence of extracellular Ca²⁺ abolished the ATP-induced decrease in Cl and K content. Alloxan (1.25 mM), an adenylate cyclase inhibitor, had an inhibitory effect on the response to ATP. The decrease in K, but not in Cl, content in the cells elicited by ATP was blocked by prior incubation with 100 ng/ml pertussis toxin, indicating the coupling of ATP to pertussis toxin-sensitive G-proteins. In conclusion, both Ca²⁺- and cAMP-dependent Cl^- permeability is present in primary cultures from duct cells of human sweat gland. The response to ATP can be mediated both by Ca²⁺- and by cAMP-dependent pathways, and is coupled to pertussis toxin-sensitive G-proteins.

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Our goal is to assess the viability of an in vitro preparation of bovine ciliary body /epithelium (CBE) in a small volume Ussing-type chamber. A new small volume Ussing-type chamber with continuous perfusion was developed for bovine CBE. The trans-CBE electrical parameters were monitored and the electrical responses of the CBE to ouabain (1 and 0.01 mM) were recorded. The trans-CBE fluxes of [¹⁴C]-L-ascorbate and [³H]-L-glucose were also studied. The bovine CBE preparation was stable inside the chamber in terms of its potential difference (PD), short circuit current (SCC) and trans-CBE resistance. They were - 0.51 ± 0.05 mV (aqueous side negative), - 5.43±0.04 μAcm^-2 and 94±2 Ω.cm² (mean±s.c.m., n=35), respectively. The preparation hyperpolarised when 0.01 mM ouabain was administered to the aqueous side, depolarised when ouabain was applied to the stromal side. [³H]-L-glucose diffusion was about 74 nEq h^-1cm^-2 in either direction (n = 12). Taking the area magnification factor of the CBE into consideration, the diffusional L-glucose flux across the bovine CBE was comparable to other tight epithelia. A significant net ascorbate flux (0.26±0.05 nEq h^-1cm^-2, n=4, p<0.01) was found in the stroma to aqueous direction. We have developed a viable in vitro bovine CBE preparation which was (1) electrically stable, (2) responsive to ouabain, (3) tight to L-glucose diffusion, and (4) capable of actively secreting ascorbate. A net trans-CBE chloride transport (0.81±0.30 μEq h^-1cm^-2, n = 12, p = 0.01) from stromal to aqueous side was found in the present in vitro model under short-circuited conditions.

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We examined alterations in cell morphology and expression of adhesion molecules in response to a general protein kinase inhibitor K252a treatment of non-adherent colon adenocarcinoma Colo201 cells. K252a induced rapid cell adhesion and spreading with concomitant formation of actin stress fibers. A protein kinase A inhibitor KT5720 also induced cell adhesion, but the rate of spread was slower than that seen with K252a. These adhesions were mediated by integrin molecules since cell adhesion required Mg²⁺, Mn²⁺ or Ca²⁺, and was inhibited by monoclonal antibodies for integrins α2 and β1. Indirect immunofluorescence microscopic observations revealed that integrin α₂ and β₁ molecules in K252a-treated cells were concentrated at sites of focal adhesion, but expressions of integrin molecules were not modulated. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin increased during K252a- or KT5720-induced cell adhesion. Immunosuppressants FK506 and cyclosporin A suppressed the K252a-induced cell adhesion and abolished tyrosine phosphorylation of cellular proteins including FAK and paxillin. Furthermore, W7 and calmidazolium, inhibitors of calmodulin, also inhibited the cell adhesion. Based on findings that FK506 and cyclosporin A are inhibitors of the calcium calmodulin-dependent protein phosphatase, calcineurin, this phosphatase may regulate integrin-dependent cell adhesion and spread of Colo201 cells. This Colo201 cell model provides a pertinent system for studying molecules involved in signal transduction pathways and can shed light on mechanisms of metastasis and invasion of colon carcinoma cells.

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Treatment with 5 μM of saikosaponin (SS) b₂ for 30 days was found to induce differentiation of B16 melanoma cells, with potentiation of expressions of melanogenesis and tyrosinase. To explore the mechanism of this effect, we observed the cell growth, cell cycle and morphology, and found that SSb₂ did not affect any of these parameters. That is, SSb₂ induced the differentiation of B16 melanoma cells without growth inhibition or cytotoxicity under conditions of low dose and long-term treatment. Phorbol ester, a protein kinase C (PKC) activator, markedly inhibited the expressions of melanogenesis and tyrosinase in both the control B16 melanoma cells and the long-term treated B16 melanoma cells. Down-regulation of the PKC activity may be involved in the effects of SSb₂.

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The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subset of axonemal inner-arm dyneins, has recently been shown to lack conventional actin owing to a serious mutation in its gene. It lacks inner-arm dyneins probably because actin is an essential subunit for their assembly. In addition, male gametes of ida5 are unable to produce the fertilization tubule, a structure that contains a core of actin filament bundles. To establish that those observed deficiencies are solely attributable to the loss of actin, and to provide a basis for future studies on the actin function in this organism, we examined in this study whether transformation of this mutant with cloned actin genes can rescue the mutant phenotypes. Cotransformation of the double mutant ida5arg2 with the wild-type actin gene and arginino-succinate lyase gene that suppresses the arg2 mutation yielded several transformants that displayed increased motility. All of them were found to have acquired the introduced actin gene in the genome and the product actin in the flagella, and regained the missing inner-arm dyneins and wild-type motility. In addition, most transformants also became able to grow the fertilization tubule when mating reaction was induced. In addition to the wild-type actin gene, we also used a chimeric actin gene in which the N-terminal 12 ammo-acid sequence of Chlamydomonas actin was replaced by that of the greatly divergent Tetrahymena actin. Transformants with this gene also resulted in recovery of inner-arm dynein and 70-80% of the wild-type level of motility. These results established that the lack of inner-arm dynein and the fertilization tubule in ida5 are consequences of its loss of conventional actin. Furthermore, they demonstrate that Chlamydomonas offers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.

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By immunofluorescence microscopic observation, monoclonal and polyclonal antibodies against a synthetic actin C-terminal peptide were found to stain too colloguial, ambiguous punctuate structures distributed throughout the cytoplasm of 3Y1 cells, independently of actin stress fibers. Antibody against rab5, a small GTP binding protein of the sorting endosome, and anti-actin antibody co-stained these punctuate structures. On the other hand, transferrin receptor, a well characterized maker of the sorting and recycling endosomes, colocalized with actin on the vesicular structures at the cell peripheral region but not at the perinuclear area where the recycling endosome localized. These observations suggest that actin molecules localize on the sorting endosomes. Tropomyosin, F-actin binding protein, also colocalized with actin on the sorting endosomes. From these results, we proposed that actin-filaments with tropomyosin constitute the membrane skeleton on the sorting endosome surface. This article is the first report to show that actin-filaments localize on the intact endosomes.

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Addition of hemin to the nuclear reconstitution system of Xenopus interphase egg extract using sperm head chromatin resulted in abnormal pseudonuclei exhibiting flattened membrane patches randomly distributed both on the surface and inside the nuclei. The structures that resembled nuclear pores were observed on these flattened membrane patch structures. Although the nucleosome structure was formed as revealed by the micrococcal nuclease digestion, the B-type lamin uptake into the nuclei was inhibited by hemin. Using heminagarose affinity chromatography, we isolated several hemin-binding proteins from fully reconstituted pseudonuclei. Some of the hemin-binding proteins bound concanavalin A (Con A). Comparison of hemin-binding proteins with those isolated from both fractions of supernatant and pellet separated by high speed centrifugation of the egg extract showed that the hemin-binding proteins of pseudonuclei were supplied from both fractions. The uptake of nuclear hemin-binding proteins did not occur in the incompletely reconstituted nuclei resulting from addition of excess sperm chromatin to the system. These results suggest that the hemin-binding proteins participate in the late steps of nuclear reconstitution during formation of the nuclear envelope.

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A novel lectin derived from silkworm faeces, named NUE, activates phagocytosis of mouse peritoneal macrophages (9). At this time, significant morphological changes of the cells take place. NUE-treated macrophages formed projection-like neurocytes within 12 hr of treatment, and appeared somewhat flat in shape with the activation of cell growth. Associated with the morphological changes, actin was organized in dot-like structures corresponding to cell-substratum contact sites in NUE-treated macrophages. Vinculin, a cytoskeletal protein involved in microfilament-membrane interaction, formed doughnut-like rings matching the actin-dots, called "podosomes". Furthermore, observation by interference reflection microscopy showed that NUE-treated macrophages adhered more strongly to the substratum at local areas. According to these changes, more proteins associated with cell-substratum contact sites became to detergent-resistant. It was shown that NUE changed adhesive form of mouse peritoneal macrophages structurally and qualitatively.

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