Tyrosine phosphorylation as a convergent pathway of heterotrimeric G protein- and rho protein-mediated Ca2+ sensitization of smooth muscle of rabbit mesenteric artery.

1. The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5'-O-(3-thiotriphosphate) (GTP(gamma)S) and receptor agonists on beta-escin-skinned smooth muscle of rabbit mesenteric artery. 2. In the homogenate of the beta-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [32P]-ADP-ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti-rho p21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTP(gamma)S-induced Ca2+ sensitization, although the effect of GTP(gamma)S at higher concentrations (> or = 30 microM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca2+-induced contraction was not affected by C3 pretreatment. 3. The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13-dibutyrate of the Ca2+-induced contraction. However, these PKC inhibitors had no effect on GTP(gamma)S- and receptor agonist-induced Ca2+ sensitization. 4. The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTP(gamma)S of the Ca2+-induced contraction without affecting this Ca2+ contraction. The inactive genistein analogue daidzein did not modify the effect of GTP(gamma)S. The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors. 5. These results suggest that rho p21 predominantly mediates GTP(gamma)S-induced Ca2+ sensitization of beta-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein-coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein- and heterotrimeric G protein-mediated Ca2+ sensitization mechanisms.

Different regulation of myofilament Ca2+ sensitivity in beta-escin-skinned cardiac and vascular smooth muscles.

We compared the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS, an activator of G-protein), phorbol 12,13-dibutylate (PDB, an activator of protein kinase C) and pimobendan (an inotropic agent with Ca2+-sensitizing action) on the Ca2+ sensitivity of the contractile proteins in beta-escin-skinned muscle preparations obtained from rabbit left ventricles and mesenteric arteries. After the skinning procedure, when GTPgammaS (100 microM) or PDB (1 microM) was added to the Ca2+ solutions, pCa50 were significantly increased in preparations obtained from vascular smooth muscle, but not from cardiac muscle, indicating that G-protein- and protein kinase C-mediated direct Ca2+ sensitization may occur only in smooth muscle, but not in cardiac muscle. In contrast, pimobendan (50 microM) increased the Ca2+ responsiveness only in cardiac muscle. Therefore, we conclude that, in addition to the common regulatory factors affecting Ca2+ sensitivity such as intracellular pH and phosphorylation by protein kinase A, there are other means of regulation of Ca2+ sensitivity working differently in cardiac and in vascular smooth muscles.

Dietary protein peptic hydrolysates stimulate cholecystokinin release via direct sensing by rat intestinal mucosal cells.

We previously demonstrated that a peptic hydrolysate of guanidinated casein strongly stimulates exocrine pancreatic secretion in chronic bile-pancreatic juice-diverted rats and cholecystokinin (CCK) release from dispersed rat intestinal mucosal cells. These results reveal that the chemically modified protein hydrolysate stimulates CCK secretion and increases pancreatic secretion by a luminal trypsin-independent direct action on the small intestine. In the present study, we examined the direct effect of peptic hydrolysates of naturally occurring dietary proteins, casein, soybean protein isolate (SPI), egg white, and wheat gluten on CCK release under in vitro trypsin-independent conditions. All protein hydrolysates significantly stimulated CCK release from dispersed rat intestinal mucosal cells. Among the hydrolysates treated, SPI hydrolysate was the most effective in stimulating CCK release. The potential of SPI hydrolysate to stimulate CCK release was increased by long-term peptic digestion. However, an SPI-like amino acid mixture did not effect CCK release. In conclusion, peptic hydrolysates of commonly ingested dietary proteins stimulate CCK release via trypsin-independent direct sensing by intestinal mucosal cells.

A case of gallbladder cancer associated with a common bile duct neuroma, and a cystic lesion of the liver with histologic findings similar to those of an inflammatory pseudotumor.

We report a rare case of gallbladder cancer associated with a common bile duct neuroma, and a cystic liver lesion with histologic findings similar to an inflammatory pseudotumor, in a patient who had had no previous abdominal surgery. The patient was a 62-year-old man whose major complaint was fever. Ultrasonography and a computed tomography scan revealed gallstones, an elevated lesion in the gallbladder, and a cystic liver lesion. Endoscopic retrograde cholangiopancreatography demonstrated stenosis of the common bile duct. Cultures of the cystic fluid and gallbladder bile were positive for Staphylococcus aureus. The patient underwent hepatectomy (inferior S4, S5, and S6), cholecystectomy, resection of the common bile duct, and right hemicolectomy. The resected specimens revealed gallbladder cancer with the microscopic appearance of a papillary adenocarcinoma, and a 12 x 4.5 x 3.5 cm cystic liver lesion with a wall 7 mm thick. Histologic studies of the wall of the cystic liver lesion revealed infiltration by histiocytes and plasma cells, and the presence of fibrous connective tissue, which findings are characteristic of inflammatory pseudotumors. A 9 x 6 mm elevated lesion, with the microscopic appearance of a neuroma, was resected from the common bile duct.

Gallbladder cancer associated with cholesterosis.

We report herein two cases of carcinoma in situ of the gallbladder associated with cholesterosis. The patient in case 1 was an 81-year-old man who underwent a cholecystectomy for cholelithiasis. The resected specimens revealed gallbladder cancer in the fundus which was diagnosed histologically as mucinous carcinoma. Other findings included 13-mm, 12-mm, and 5-mm polypoid lesions in the neck of the gallbladder which macroscopically appeared to be cholesterol polyps, but histologically demonstrated carcinoma in situ with cholesterosis. The patient in case 2 was a 76-year-old man in whom ultrasonography revealed a highly echogenic, elevated lesion in the gallbladder. Cholecystectomy was performed, and a 33 x 28-mm papillary, elevated lesion with cholesterosis was resected from the neck of the gallbladder. Histologically, this was demonstrated to be papillary adenocarcinoma in situ with cholesterosis surrounded by glandular dysplasia. The distribution of the carcinomas and cholesterosis in both of these patients suggests that the adenoma or carcinoma of the gallbladder had occurred first. Then, the tumor epithelium absorbed cholesterol from the bile, and foamy cells were produced. Thus, when treating cholesterol polyps, it should be remembered that it is often difficult to distinguish between cholesterol polyp and gallbladder cancer with cholesterosis.

Cardiac profile of EGIS-9377, a novel cardiotonic agent as a Ca2+ sensitizer with bradycardiac activity.

The cardiac profile of EGIS-9377 ¿2-(1-methylthio)-5-(2-morpholinoethylamino)-8,9-dihydro-7H-thi opyrano[3,2-d][1,2,4]triazolo[1,5-a]pyrimidine dihydrochloride¿, a newly synthesized cardiotonic agent, was compared with those of pimobendan and isoprenaline in cardiac preparations isolated from guinea pigs. The positive inotropic potency and efficacy of EGIS-9377 were equal to those of pimobendan in electrically paced papillary muscles, with each agent maximally increasing force of contraction by 30-35% of the maximum effect of isoprenaline. The positive inotropic effects of EGIS-9377 and pimobendan were accompanied by an increase in the relaxation time of the isometric contraction curve, whereas that of isoprenaline was associated with an abbreviation of this parameter. Pimobendan significantly increased the spontaneously beating frequency of right atria, and its positive chronotropic effect amounted to 40% of the maximum effect of isoprenaline. In contrast, EGIS-9377 exerted a significant negative chronotropic action, which resulted in a 30% decrease in the basal frequency. In beta-escin-skinned trabecular muscles, both EGIS-9377 and pimobendan substantially enhanced contractions induced by Ca2+. EGIS-9377 at concentrations to cause a significant negative chronotropic action produced a marked prolongation of action potential duration (APD) in guinea pig papillary muscle and greatly inhibited the delayed rectifier potassium current (I(K)) in guinea pig ventricular single cells. This suggests that the negative chronotropic effect of EGIS-9377 may, at least in part, be due to the prolongation of APD as a result of the I(K) inhibition. The present results indicate that EGIS-9377 efficiently increases myocardial contractile force possibly due to its Ca2+ -sensitizing activity, and yet produces a substantial negative chronotropic action. This cardiac profile of EGIS-9377 is suggested to be a clinically favorable feature compared with the inotropic agents having cyclic AMP generation or phosphodiesterase inhibition as their action mechanisms.

Short-chain fatty acids enhance diffusional ca transport in the epithelium of the rat cecum and colon.

We examined the effect of short-chain fatty acids (SCFAs) on Ca absorption from the large intestine in rats in vitro. An Ussing-type chamber technique was used to determine the net transport of Ca from the luminal side to the basolateral side of isolated epithelium in cecum and colon preparations. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. Both solutions were warmed at 37 degrees C and bubbled with 95% O2 and 5% CO2. During and after the incubation period (30 min or 60 min), the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was evaluated. The addition of 80 mM acetic acid, 40 mM propionic acid and 10 mM butyric acid to the mucosal medium increased net Ca absorption (about 300%) in the cecum and colon. An individual application of acetic, propionic or butyric acid (0.01 to 100 mM) to the mucosal medium also increased net Ca absorption at doses of 10 mM and /or 100 mM in the cecum and colon. An increase in solute concentration in the mucosal medium by addition of glycerol or PGE400, or a decrease in pH (7.0-3.0) by addition of HCl did not affect transepithelial Ca transport. We concluded that SCFAs affect the epithelial tissue and promote Ca absorption from the large intestine in vitro. The enhancement of Ca transport induced by SCFAs might be involved in the paracellular transport mechanism.

Tetrocarcins, novel antitumor antibiotics. I. Producing organism, fermentation and antimicrobial activity.

A novel antitumor antibiotic complex has been obtained from the culture broth of Actinomycete strain DO-11 (KY11091) isolated from a soil sample collected in Sendai, Miyagi, Japan. On the taxonomic studies the producing organism is described as Micromonospora chalcea KY11091. For the production of the antibiotics, soluble starch served as a good carbon source and yeast extract was the best nitrogen source tested. The antibiotic complex designated as tetrocarcins is active against Gram-positive bacteria, but is not active against Gram-negative bacteria. Tetrocarcin A showed bacteriocidal activity against Bacillus subtilis.

Gilvocarcins, new antitumor antibiotics. 5. Biosynthesis of gilvocarcins: incorporation of 13C-labeled compounds into gilvocarcin aglycones.

The biosynthesis of the aglycones of gilvocarcins V and M has been studied with 13C-labeled precursors. The aglycones have been shown to be formed via the acetate pathway and a route for their biosynthesis is proposed which involves secondary addition of an alkyl group.

DC-52, a novel antitumor antibiotic. 2. Isolation, physico-chemical characteristics and structure determination.

DC-52, C18H22N2O4, is a new antitumor antibiotic produced by Streptomyces melanovinaceus nov. sp. The structure of DC-52 has been determined by consideration of spectral data. It has the novel skeleton, 8,11-iminoazepinoisoquinoline.

Uptake of 2'-amino-2'-deoxyguanosine by Escherichia coli and its competition by guanosine.

The antibacterial activity of a new nucleoside antibiotic, 2'-amino-2'-deoxyguanosine (2AG), is reversed by guanosine and other purine nucleosides. 2AG is apparently taken up by E. coli by a mechanism different from that of guanosine; guanosine inhibits this uptake non-competitively. Insensitive E. coli strains and the resistant mutant obtained from the sensitive strain also took up 2AG.

Mechanism of growth inhibition by 2'-amino-2'-deoxyguanosine in Escherichia coli.

The mechanism of inhibition of Escherichia coli by the new nucleoside antibiotic, 2'-amino-2'-deoxyguanosine (2AG), is described. Upon the addition of 2AG, the syntheses of macromolecules continued for 15 minutes. After this lag time, protein synthesis sharply decreased, Rna synthesis slightly decreased, but DNA synthesis was not affected. Tritiated 2AG was readily incorporated into the acid-soluble fraction of cells in the form of the mono-, di- and triphosphates. In the acid-soluble fraction, radioactivity was found only in the RNA fraction. The major part of the radioactivity was found to be guanylate; only 25% existed as the nucleotide of 2AG. In resistant strains of E. coli, there was a lower degree of phosphorylating activity and less incorporation of 2AG into RNA per unit of cell mass increase. These results suggest that 2AG inhibits growth by its incorporation into RNA and the subsequent distrubance of RNA function causing a block in protein synthesis.

DC-52, a novel antitumor antibiotic. 1. Taxonomy, fermentation and biological activity.

A novel antitumor antibiotic, DC-52 was found in the culture broths of Actinomycete DO-52. The producing organism was subsequently determined to be a new species and named Streptomyces melanovinaceus nov. sp. For the production of the antibiotic, soluble starch served as a good carbon source and soybean meal was a good nitrogen source tested. The antibiotic DC-52 is active against Bacillus subtilis, Staphylococcus aureus and Klebsiella pneumoniae, but not active against most Gram-negative bacteria. The antibiotic is also active against mouse leukemia P388.

Gilvocarcins, new antitumor antibiotics. 4. Mode of action.

The mode of action of gilvocarcins was studied. Gilvocarcins V, M and A possessed antibacterial activities decreasing in that order. Gilvocarcin V inhibited DNA synthesis in Bacillus subtilis through strong interaction with DNA and resulting cleavage. Gilvocarcin M showed interaction with DNA and a small change in DNA mobility upon electrophoresis in agarose gel, while gilvocarcin A showed no interaction with DNA, thus reflecting their relative biological activities.

DC-86-M, a novel antitumor antibiotic. I. Taxonomy of producing organism and fermentation.

A novel antibiotic, DC-86-M was isolated from the culture broth of a new isolate, DO-86, from the soil sample collected in Machida-shi, Japan. The producing organism was found to belong to Streptomycetes, for it formed aerial mycelia and chains of spores and its cell wall analyses revealed the presence of LL-diaminopimelic acid. The morphological, cultural and physiological characteristics of the strain DO-86 resemble closely those of Streptomyces luteogriseus and we concluded that the strain DO-86 could be designated as Streptomyces luteogriseus DO-86. The antibiotic was produced in the fermentation medium consisting of lactose 20 g, glucose 10 g, Pharmamedia 15 g, yeast extract 5 g, meat extract 10 g and CaCO3 2 g per liter of tap water.

DC-86-M, a novel antitumor antibiotic. II. Structure determination and biological activities.

A novel antibiotic, DC-86-M was isolated from the culture broth of Streptomyces luteogriseus DO-86. The antibiotic has the molecular formula of C17H14N2O5 and belongs to the phenazine antibiotics. Its structure has been elucidated by mass and NMR spectra. It is active against Gram-positive and Gram-negative bacteria and experimental murine sarcoma 180.

CV-1, a new antibiotic produced by a strain of Streptomyces sp. I. Fermentation, isolation and biological properties of the antibiotic.

A new antibiotic, CV-1, was isolated from the culture broth of a Streptomyces sp. by various chromatographies. CV-1 showed antibacterial activity against Escherichia coli in cooperation with spiramycin, a macrolide antibiotic. The mode of action of CV-1 seemed to be the inhibition of lipopolysaccharide synthesis.

Gilvocarcins, new antitumor antibiotics. 2. Structural elucidation.

Gilvocarcin V(1), C27H28O9, m.p. 264 approximately 267 degrees C (dec.), and gilvocarcin M(2), C26H26O9, m.p. 245 approximately 248 degrees C (dec.), are new antitumor antibiotics produced by Streptomyces gilvotanareus. The structure of gilvocarcins has been determined by chemical degradation, nmr and mass spectra. They have a benzonaphtopyran-one system, to which the furanose moiety is linked through a C-C glycosyl bond.

Gilvocarcins, new antitumor antibiotics. 3. Antitumor activity.

Gilvocarcin V, isolated rom a Streptomyces culture showed activity against experimental tumors such as sarcoma 180, Ehrlich carcinoma, Meth 1 fibrosarcoma, MH134 hepatoma and lymphocytic leukemia P388. In particular, 40% of treated mice survived for 60 days, after intraperitoneal administration of gilvocarcin V to mice bearing Ehrlich ascites carcinoma. But it was marginally active against B16 melanoma and did not produce prolongation of lifespan of mice bearing Lewis lung carcinoma.

Tetrocarcins E1, E2, F and F-1, new antibiotics. Fermentation, isolation and characterization.

New components of tetrocarcins (E1, E2, F and F-1) were found in the culture broth of Micromonospora chalcea KY 11091 that was known to produce tetrocarcins A, B and C. Tetrocarcin F-1 consisted of tetronolide and nitro sugar (tetronitrose). Tetrocarcins E1 and E2 consisted of F-1 and deoxy sugar (L-digitoxose). Tetrocarcin F consisted of F-1 and two deoxy sugars (their structures were not yet determined). They all showed antibacterial activities against Gram-positive bacteria and the specific activity decreased with decrease in the numbers of deoxy sugars attached to the aglycone.