Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

Estimation of Type-Specific Neutralizing Antibody to Herpes Simplex Virus Type 2 in Uterine Cervical Cancer Patients by a New Absorption Method

A simple method of estimating type-specific neutralizing antibody to type 2 herpes simplex virus (HSV-2) was devised with the use of the microneutralization system. Serially diluted serum was mixed in the well with a constant amount of type 1 virus (HSV-1), and after 3 days' incubation at 37 C, the plate was irradiated with ultraviolet light. The absorbing HSV-1 consisted of culture fluid plus an extract of infected Vero cells not especially concentrated. The well then received indicator HSV-1 or HSV-2, and after being left at 37 C for 1 hr a suspension of dispersed Vero cells was dropped into the wells, following our standard neutralization procedure. Preliminary tests with rabbit antisera showed that even a low level of HSV-2 antibody was detected by this method, unless an exceptionally high titer of HSV-1 antibody originally coexisted with the HSV-2 antibody. Sera from acutely infected persons testified to the specificity of the antibody so detected. It was revealed by means of the new technique that the rate of HSV-2 antibody was significantly higher in uterine cervical cancer patients than in control women. There was no correlation between the clinical stage of cervical cancer and the presence of HSV-2 antibody.     

The Effects of Ethylene on the Coordinated Synthesis of Multiple Proteins: Accumulation of an Acidic Chitinase and a Basic Glycoprotein Induced by Ethylene in Leaves of Azuki Bean, Vigna angularis

The ethylene-induced synthesis and accumulation of proteinswere studied in the primary leaves of azuki bean (Vigna angularis).Seven different proteins, designated AZ17, 23, 27, 32, 35, 36,42 according to their molecular masses, were synthesized andaccumulated in response to ethylene. AZ27 and AZ42 were purifiedto homogeneity and characterized. AZ27 was identified as anacidic chitinase and accumulated in the extracellular space.The sequence of the 40 N-terminal amino acids of AZ27 showedno similarity to that of a basic chitinase from bean and tobacco,but it was highly homologous to that of a chitinase from virus-infectedcucumber leaves. AZ42 was identified as a glycoprotein thataccumulated intracellularly. A search for proteins with sequenceshomologous to an internal sequence of 18 amino acids in AZ42was unsuccessful. Immunochemical examination revealed that auxinand abscisic acid enhanced the ethylene-induced accumulationof AZ27 but not of AZ42. In contrast, levels of AZ42 were notaffected by auxin or abscisic acid, but cytokinin suppressedthe accumulation of one of the doublet bands of AZ42. TranslatablemRNAs coding for AZ27 and AZ42 were not present in leaves thathad not been treated with ethylene, but levels of these mRNAsincreased after such treatment. (Received March 1, 1991; Accepted May 8, 1991)     

Stimulation of anaphylaxis in the mouse footpad by dietary fish oil fatty acids

The effect of fish oil-derived omega-3 (omega-3) fatty acids on anaphylaxis, Arthus and delayed type hypersensitivity reactions in mice has been investigated. Mice on a normal chow diet were fed eicosapentaenoic acid and docosahexaenoic acid at a dose of 500 and 333 mg/kg/day, respectively, by a gastric tube over a period of 61 days. Control groups were given water, safflower oil or oleic acid. Anaphylactic and Arthus type reactions were induced in the mouse footpad using bovine serum albumin as an antigen. Carrageenin was utilized to produce a delayed type hypersensitivity reaction. The animals fed omega-3 fatty acids induced a more anaphylactic foodpad reaction. There was no significant effect of the diet on Arthus and delayed type hypersensitivity responses. There was no effect of the fish oil-supplemented diet on production of antibodies to bovine serum albumin. Synthesis of prostaglandin E2 by peritoneal macrophages was significantly inhibited in the animals fed omega-3 fatty acid-enriched fish oil, while leukotriene B4 production was not affected. These results suggest that a diet enriched in omega-3 fatty acids modulates production of arachidonic acid metabolites and this may influence anaphylaxis, but not Arthus and cellular mediated hypersensitivity responses.     

Inhibition of Auxin-Stimulated Elongation of Cells in Rice Lamina Joints by a Feruloylated Arabinoxylan Trisaccharide

A feruloylated arabinoxylan trisaccharide inhibited IAA-stimulatedelongation of cells in rice lamina joints. The de-esterifiedcompound, an arabinoxylan trisaccharide, did not inhibit suchelongation. This is the first report that feruloylated arabinoxylanfragments are involved in the regulation of plant growth. (Received September 18, 1991; Accepted January 13, 1992)     

Analysis of tegumental surface proteins of Schistosoma mansoni primary sporocysts

Axenically transformed primary sporocysts of Schistosoma mansoni (NMRI strain) were labeled with 125I in an effort to identify sporocyst proteins exposed at the tegumental surface. Using the 125I activating reagent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril, in conjunction with SDS-PAGE and autoradiography, up to 12 bands were radiolabeled out of 60 components visualized by silver staining. Labeled proteins ranged in apparent Mr from greater than 200 to less than 12 kDa. Pronase treatment of living sporocysts after radioiodination removed all labeled material, suggesting that only surface proteins were being iodinated. Western blot analysis employing 5 monoclonal antibodies (MAB's) to sporocyst surface antigens revealed a wide range of reactivities which produced banding patterns closely reflecting autoradiograms of identical samples. The concomitant removal by Pronase of immunoreactive and radiolabeled surface proteins with identical Mr in the range of 90-130 kDa suggests that epitopes recognized by these antibodies are associated with these higher molecular weight surface proteins. However, although Pronase removes all labeled surface proteins, substantial nonradiolabeled, immunoreactive material with Mr less than 90 kDa still remains on enzyme-treated parasites. This indicates that MAB-reactive epitopes, in addition to their occurrence with surface proteins, are also associated with either unlabeled, protease-resistant surface components or internal antigens. The immunohistochemical localization of antibody-reactive material in gland-like structures within sporocysts supports an internal source for nonradiolabeled, immunoreactive components. Finally, the periodate sensitivity of the epitopes recognized by all tested MAB's suggests that carbohydrate moieties may represent a common and extremely immunogenic constituent of the sporocyst surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.