Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord

The binding site for calcitonin gene-related peptide (CGRP) was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in an active form from porcine spinal cord. 125I-labeled human alpha-CGRP (125I-CGRP) binding to the solubilized protein was determined by filtration using a GF/B glass filter. The maximal binding activity (approximately 60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I-CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.     

Effects of Prolonged Depolarization on the Nicotinic Acetylcholine Receptors of PC12 Cells

To determine whether prolonged depolarization and/or changes in intracellular Ca2+ concentrations stimulate adaptive responses of neuronal nicotinic acetylcholine receptors, PC12 pheochromocytoma cells were grown in medium containing various concentrations of K+. Nicotinic receptor function was determined as carbachol-stimulated uptake of 86Rb+. Cells were exposed to 50 mM K+ for up to 4 days and then allowed to repolarize for 60 min. Under these conditions, no changes in basal or carbachol-stimulated uptake of 86Rb+ were observed. Furthermore, neither the time course of carbachol-stimulated uptake or the carbachol concentration dependence of 86Rb+ uptake was altered. Finally, concurrent depolarization did not affect the functional down-regulation produced by chronic exposure of the cells to carbachol. Thus, neuronal nicotinic acetylcholine receptors on PC12 cells do not appear to be regulated by depolarization or prolonged elevation of the intracellular Ca2+ level.     

Effects of environmentally hazardous chemicals on the emergence and early growth of selected Australian plants

The effect of soil-incorporated copper, tri-allate, and anthracene on the emergence and early growth of three Australian native species (Banksia ericifolia, Casuarina distyla andEucalyptus eximia) and three crop species (Avena sativa, Cucumis sativus andGlycine max), was assessed using OECD Test Guideline 208. The crop species are sensitive species used in overseas phytotoxicity testing, and their responses were compared with those of the native species. Seeds were grown in pots in a glasshouse in a sandy loam soil at the chemical concentrations of 0, 10, 100, 1000 and 2000 mg kg^–1. LC50 and EC50 values were determined for each species. The most sensitive species was the monocotyledonA. sativa, while among the five dicotyledonsC. distyla was most sensitive. All three chemicals delayed emergence and affected seedling growth. The results indicate that the conditions of the OECD Test Guideline can be met under Australian conditions, but that the Guideline requires modification for use with Australian native species.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Protein Kinase C and Melanocyte Transformation

Melanoma cells which have been isolated from metastatic melanoma tissue are able to survive and proliferate in serum supplemented media. In contrast, normal human melanocytes require the presence of growth stimulators if they are to survive in culture. A tumor promotor, 12–0-tetradecanoyl-phorbol-13-acetate (TPA) and substances that increase intracellular levels of cyclic-adenosine-monophosphate (cAMP), such as cholera toxin or isobutylmethyl xanthine, have been widely used for this purpose. The phorbol diester receptor was shown in 1982 to be the phospholipid- and calcium-dependent enzyme protein kinase C (PKC). We therefore directed our studies to the role of PKC regulation in the growth of normal human melanocytes and their transformation. Our studies show that while melanoma cells are inhibited by TPA, the growth of normal melanocytes is stimulated in a dose-dependent manner. The inhibitor, 1-(5-isoquinolinesulfonyl)-2-methyl-piperizine dihydrochloride (H7), which has been found to be the most specific for PKC, had no effect on the growth of normal melanocytes, but inhibited the growth of melanoma cells in a dose-dependent manner. PKC was isolated from the membrane and cytosol of normal melanocytes and melanoma cells. The basal (resting) levels of PKC activity in normal melanocytes was low compared to that measured in melanoma cells, and after short-term (1 hour) treatment with TPA the PKC activity was greatest at the membrane, with the activity decreasing the cytosol. Upon prolonged (48 hours) treatment with TPA, this redistribution of activity continued in normal melanocytes and the total activity increased. In melanoma cells, however, the total PKC activity decreased, particularly in the membrane fraction. A difference in activity and distribution of the enzyme was also seen after short-term (1 hour) treatment with H7. There was very little effect seen on PKC in normal melanocytes; however, the effect on melanoma cells was similar to that seen after 48 hours of exposure to TPA with a decrease in total activity, particularly in the membrane fraction. These results indicate that the regulation of PKC, in particular its activation by TPA, is altered during the transformation of normal human melanocytes     

TPA对原代白血病细胞的诱导分化作用

本文报告了TPA对32例不同类型白血病细胞的体外分化诱导结果。TPA(1.6×10~-7M)可诱导急性非淋巴细胞(ANLL)白血病细胞迅速出现单核巨噬细胞分化标志:细胞贴壁、胞浆丝状伪足形成,具有类似巨噬细胞的形态改变及相应的细胞化学反应特征。急性淋巴细胞白血病(ALL)和桨细胞白血病(PCL)细胞不发生上述变化,表现为细胞聚集成闭现象。慢性淋巴细胞白血病(CLL)出现桨细胞样形态转化。初发与复发病例的诱导反应相类似。TPA体外诱导分化实验,有助于了解病人白血病细胞的分化潜能,对于鉴别粒单系和淋巴系两类白血病,尤其对于用常规方法分型困难的低分化白血病有一定的临床诊断意义。     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.     

云南省(虫齿)目二新种和灰背蛇(虫齿)的记述((虫齿)目:厚(虫齿)科、叉(虫齿)科、围(虫齿)科)

本文记述了云南省(虫齿)目二新种,Tapinella bannana sp.n.和Peripsocus plurimaculatus sp.n.及一新种记录种Ophiodopelma semicets Lee and Thornton,其雄虫为首次记载。     

Opioid receptor antagonist affinity ligands: 6β-bromoacetamido-6-desoxynaltrexone and 6β-thioglycolamido-6-desoxynaltrexone

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [³H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had K_i values of 1×10^–9 and 1×10^–10 M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5×10^–8 and 1×10^–8 M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [³H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1×10^–7 M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED₅₀ dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.     

Sigma receptors in schizophrenic cerebral cortices

Antipsychotics represent high affinity for sigma receptors and sigma-like drugs often have the psychotomimetic properties. Besides, the receptors are unevenly distributed in human brain. These findings suggest that sigma receptors might be involved in the pathophysiology of schizophrenia. Sigma receptors in rat and human brain were measured with [³H]-1, 3, di-o-tolylguanidine (DTG) and non-specific binding of [³H]DTG was determined in the presence of 10^–5M haloperidol. Monovalent and divalent cations strongly inhibited [³H]DTG binding. Glutamate, aspartate and glycine also decreased the binding to human cerebral membranes. With post-mortem brain samples from 12 schizophrenics and 10 controls, sigma receptors were measured in 17 areas of cerebral cortex. Sigma receptors binding showed the regional differences in the cortex, but no significant differences between schizophrenics and controls were observed except the superior parietal cortex where the binding significantly increased in the schizophrenic group. These results suggest that sigma receptors in cerebral cortices might not be directly concerned with the pathophysiological role in schizophrenia.Dedicated to Dr. Morris Aprison. Received too late for publication in special issue.