Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression

Vascular smooth muscle cells (VSMC) in the rat mesenteric artery show specific immunofluorescent staining with antisera against purified human uterine myosin (ASMM) but not human platelet myosin (APM). However, in primary cultures produced by enzymatic dissociation of this vessel, VSMC stain specifically with both ASMM and APM within 5 h after plating and throughout growth to confluence (4-10 d). In confluent cultures, APM staining remains bright while ASMM staining is reduced in intensity in most cells. In contrast, cellular myosin content, determined by quantitative SDS PAGE, is comparable in confluent and growing cultures. Immunoprecipitation of high salt extracts of cultured VSMC with ASMM and APM yields myosins with the same mobilities on SDS PAGE. When serial, exhaustive precipitations are performed with one antiserum, followed by reprecipitation with the other, myosin in subconfluent and confluent VSMC cultures is exhaustively precipitated by either antiserum, thus indicating complete immunological cross- reactivity. These results might be explained by synthesis of a new myosin isoform reactive with both ASMM and APM. However, the development of APM staining in cultured VSMC did not require protein synthesis. Therefore, it is more likely that the changes in immunofluorescent staining observed in vitro reflect conformational alterations, perhaps related to cytoskeletal rearrangements. These changes in myosin antigenic expression may be relevant to the problem of VSMC phenotypic modulation both in vitro and in vivo.     

Establishment of a tumor-specific immunotherapy model utilizing TNP-reactive helper T cell activity and its application to the autochthonous tumor system

Preinduction of potent hapten-reactive helper T cell activity and subsequent immunization with hapten-coupled syngeneic tumor cells result in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-hapten helper T cells and tumor-specific effector T cells. On the basis of this augmenting mechanism, a tumor-specific immunotherapy protocol was established in which a growing tumor regresses by utilizing a potent trinitrophenyl (TNP)-helper T cell activity. C3H/He mice were allowed to generate the amplified (more potent) TNP-helper T cell activity by skin painting with trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide. Five weeks later, the mice were inoculated intradermally with syngeneic transplantable X5563 tumor cells. When TNCB was injected into X5563 tumor mass, an appreciable number of growing tumors, in the only group of C3H/He mice in which the amplified TNP-helper T cell activity had been generated were observed to regress (regressor mice). These regressor mice were shown to have acquired tumor-specific T cell-mediated immunity. Such immunity was more potent than that acquired in mice whose tumor was simply removed by surgical resection. These results indicate that in situ TNP haptenation of the tumor cells in TNP-primed mice can induce the enhanced tumor-specific immunity leading to the regression of a growing tumor. Most importantly, the present study further investigates the applicability of this TNP immunotherapy protocol to an autochthonous tumor system. The results demonstrate that an appreciable percent of growing methylcholanthrene-induced autochthonous tumors regressed by the above TNP immunotherapy protocol. Thus, the present model provides an effective maneuver for tumor-specific immunotherapy in syngeneic transplantable as well as autochthonous tumor systems.     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.     

Acanthaster planci infestations of reefs and coral assemblages in Japan: a retrospective analysis of control efforts

Reef-building corals have been extensively degraded by Acanthaster planci infestations which have continued to spread throughout the Ryukyu archipelago since 1969. Intensive control efforts were undertaken by fishermen and divers by hand-collecting and disposal on land with removal of about 13 million starfish at the total cost of over 600 million yen from 1970 to 1983 fiscal year. The control programs were mostly unsuccessful for saving the reefs from predation because the efforts were executed on the basis of collecting efficiency, so that significant numbers of starfish continued predation after each belated campaign. Certain coral assemblages outside the Ryukyus were infested with unusually large numbers of A. planci simultaneously with the northern part of Okinawa Island and its neighboring islands in the early seventies. A shift of infestation sites occurred in the extratropical waters in the mid-seventies when the warm current, Kuroshio, changed its path and left the coast of Honshu, the main island of Japan. The Kuroshio is considered to be transporting larval A. planci downstream from the Ryukyus where large aggregations have continued to exist at different areas all through the period.     

Inhibitory effect of long chain fatty acyl CoAs on RNA polymerase from Escherichia coli

RNA polymerase from Escherichia coli was inhibited by long chain fatty acyl CoAs, such as myristoyl CoA (Ki = 17.2 microM), palmitoyl CoA (Ki = 8.9 microM), oleoyl CoA (Ki = 5.5 microM), and stearoyl CoA (Ki = 0.94 microM). The inhibition by these CoA thioesters was non-competitive against nucleoside triphosphates. Short chain fatty acyl CoAs, such as acetyl CoA, propionyl CoA, acetoacetyl CoA, butyryl CoA, and decanoyl CoA, failed to inhibit RNA polymerase. CoA, Na-myristate, Na-palmitate, Na-oleate, Na-stearate, palmitoyl carnitine, and carnitine did not inhibit the enzyme. The inhibition of RNA polymerase by long chain fatty acyl CoAs was competitive against template DNA.     

Vasorelaxing actions of molsidomine and its metabolites, in comparison with nitroglycerin

The effect of a novel antianginal agent, molsidomine (N-ethoxycarbonyl-3-morpholinosydnonimine) (SIN-10) and its metabolites, 3-morpholinosydnonimine (SIN-1) and N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A) on isolated dog blood vessels were investigated. SIN-1 and SIN-1A elicited a concentration-dependent relaxation of prostaglandin F2 alpha, contracted strips, while SIN-10 was without effect even in a concentration of 10(-4) M. The mean effective concentration (EC50) values of SIN-1A were much lower than SIN-1 and other vasodilators including nitroglycerin. The time course of relaxation was more rapid and transient in response to SIN-1A than to SIN-1. Adrenergic and cholinergic blocking agents did not affect the relaxing responses to SIN-1 and SIN-1A. SIN-1A also attenuated the norepinephrine-, KCl-, Ca2+-, or electrical transmural stimulation-induced contractile response, but SIN-1A increased the [3H]norepinephrine release from the adrenergic nerve terminals in response to transmural stimulation. Methemoglobin, which reportedly binds nitric oxide, or methylene blue, an inhibitor of guanylate cyclase, attenuated the relaxing response to SIN-1A. These results indicate that the vasodilating action of molsidomine results from the direct action on the vascular smooth muscle and suggest that the action is caused by its metabolites, probably SIN-1A, which contains a nitric oxide-moiety in the molecule. The possible mechanism of vasorelaxing action of SIN-1A is discussed in comparison with that of nitroglycerin.     

Effects of haloperidol and sulpiride on dopamine-induced inhibition of nucleus accumbens neurons

Microiontophoretic study was performed to elucidate dopaminergic mechanism in the nucleus accumbens (Acc) of rats anesthetized with chloral hydrate. Iontophoretically applied dopamine produced an inhibition of glutamate-induced firing in 28 (62%) out of 45 Acc neurons tested. The dopamine-induced inhibition of 14 Acc neurons was clearly antagonized by simultaneous application of haloperidol, and a partial antagonism by sulpiride was observed in 3 out of 10 Acc neurons. These results indicate that dopamine produces an inhibition of the Acc neuron and that, compared to haloperidol, sulpiride is a less potent blocker of the postsynaptic dopamine receptor involved in the dopamine-induced inhibition.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.