SYNTHESIS AND ADENOSINE RECEPTOR AFFINITY AND POTENCY OF 8-ALKYNYL DERIVATIVES OF ADENOSINE

Adenosine derivatives bearing different (ar)alkynyl chains at the 8-position were synthesized and tested at human adenosine receptors. Binding studies showed that all compounds possess affinity for the A₃ subtype in the high nM range. Moreover, guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding assay indicated that the 8-alkynyl adenosines behaved as antagonists of NECA at A₃ receptors.     

NOVEL METHOD FOR THE STUDY OF RECEPTOR Ca2+ SIGNALLING EXEMPLIFIED BY THE NK1 RECEPTOR

ABSTRACT

We have used a novel technology (NovoStar from BMG Labtechnologies) for the study of the Ca²⁺ signalling of the human tackykinin NK1 (hNK-1 receptor). The NovoStar is a microplate reader based on fluorescence and luminescence. The instrument implements a robotic pipettor arm and two microplate carriers, typically one for samples and one for cells. The robotic pipettor arm can transfer sample (agonist or antagonist) from the sample plate or other liquid containers to the cell plate, facilitating the study of Ca²⁺ signalling to such a degree that the instrument can be used for Medium Throughput Screening (MTS). Using the NovoStar we have found the molecular pharmacology of the NK1 receptor to be comparable to that observed in classical signal transduction assays. Thus, we have observed an EC₅₀ value of 3?nM for substance P induced Ca²⁺ response. This value corresponds well with previously published values for substance P induced IP and cAMP turnover. Using the NovoStar technology we have studied the pharmacological profile of the well known non-peptide NK1 receptor antagonists CP96,345 and SR140,333 in respect of inhibition of the Ca²⁺ response induced by substance P. Interestingly, the antagonistic potency of the antagonists depended greatly on the experimental design, e.g., a dependency of timing in the addition of antagonists vs. agonist was noted. Also, metal-ion site engineered NK1 receptors were tested for the ability of metal-ions to inhibit signalling. It is concluded that the NovoStar is a reliable tool for the study of receptor Ca²⁺ signalling, both as a research tool and as a MTS system.     

A NOVEL TECHNIQUE FOR PREPARATION OF AXENIC CULTURES OF SYMBIODINIUM (PYRROPHYTA) THROUGH SELECTIVE DIGESTION BY AMOEBAE1

The marine amoeba Trichosphaerium Am-I-7 was used as a tool for preparing unialgal axenic cultures of nondigestible Symbiodinium and Porphyridium species. The resistance of these unicellular algae to the amoebal digestive enzymes, and the differential digestion of bacteria, protozoans, and other algae, resulted in cleansed cells of Symbiodinium and Porphyridium that remained in the amoebal food vacuoles. During multiple fission, the amoeba evacuated its food vacuoles and released the trapped and intact algae, which were then successfully cultured. This method of cleaning was especially useful with algal species that were sensitive to antibiotics or other germicidal agents.     

A MICRO-SCALE PROCEDURE FOR THE PREPARATION OF SUBCELLULAR FRACTIONS FROM INDIVIDUAL AUTONOMIC GANGLIA

Abstract— A microscale modification for the preparation of subcellular fractions employing milligram and submilligram amounts of neuronal tissue (brain nuclei and autonomic ganglia) is described.
Electron microscope characterization and enzymic studies were carried out on the six subcellular fractions of sympathetic ganglia of cat thus prepared.
The synaptosomal preparations obtained from individual ganglia were poorer in their nerve ending content than those obtained from brain by previous investigators. The highest RSA for AChE was found in layer L₂ which was rich in membranes and vesicle components. ChAc activity was also highly concentrated in layers L₂ and L₃ (membranes, nerve ending-like particles, mitochondria and 'ghosts'). MAO activity was particularly high in the layers L₄ and L₅ which contained a large number of mitochondria. Layer L₁ (membrane fragments) and particularly layer L₆ which contained mainly collagen fibres, were low in activity of all three enzymes.
After preganglionic denervation, both ChAc and AChE activities were significantly reduced in the purest nerve ending fraction, L₃ while MAO activity was practically unchanged.     

一种从土壤样品中选择性分离假诺卡氏菌的方法

为提高假诺卡氏放线菌的分离效率,根据其营养特性和对抗生素的敏感性,设计、检验了5种选择性分离培养基;实验检测了模式菌株在不同培养基上的生长情况,结果表明培养基S1和S2对假诺卡氏菌的生长有显著的选择性。经该方法从韩国、印度尼西亚和中国广西地区不同土样中分离到一些假诺卡氏放线菌。     

小偃麦的选育及其形成途径的研究

对普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ）与中间偃麦草Ｔｈｉｎｏｐｙｒｕｍ ｉｎｔｅｒｍｅｄｉｕｍ）杂交衍生的八倍体小偃麦,硬粒小麦（Ｔｒｉｔｉｃｕｍ ｄｕｒｕｍ）与中间偃麦草杂交衍生的六倍体小偃麦的形成过程进行系统的细胞遗传学研究,并用荧光原位杂交（Ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ）技术研究了小偃麦的染色体构成。结果表明：小偃麦的形成是由杂种Ｆ１产生非减     

ADENOSINE KINASE OF MAMMALIAN BRAIN: PARTIAL PURIFICATION AND ITS ROLE FOR THE UPTAKE OF ADENOSINE

Abstract— Adenosine metabolism in the homogenate of brain mainly undergoes deamination to inosine and hypoxanthine, while uniformly labelled [¹⁴C]adenosine injected into the carotid artery or [8-¹⁴C]adenosine incubated with brain slices was mostly phosphorylated to [¹⁴C]adenine nucleotides in brain cells. Adenosine kinase has now been partially purified from homogenates of guinea pig brain. The kinase preparation was free of adenosine deaminase, almost free of adenosine triphosphatase and had a K_m of the order of 2 × 10^-5M for adenosine.
Kinetic studies with brain slices showed that adenosine reached the cells by diffusion and that the diffusion was facilitated by subsequent phosphorylation to adenine nucleotides. From the following experimental results, it is concluded that the phosphorylation is catalysed by adenosine kinase quantitatively. (1) During the uptake and phosphorylation of adenosine by brain slices, the nucleoside did not split to adenine and ribose moieties. (2) The rate of formation of adenine nucleotides in the slices was a hyperbolic function of the concentration of adenosine in the medium, showing an apparent K_m foradenosine of the order of 2 × 10^-5 M. (3) Some analogues of adenosine inhibited both the facilitated diffusion of adenosine and the kinase activity, but ouabain (0.005 mM) did not inhibit either.     

2-SUBSTITUTED PI SYSTEM DERIVATIVES OF ADENOSINE THAT ARE CORONARY VASODILATORS ACTING VIA THE A2A ADENOSINE RECEPTOR

Compound 20 (CVT-3146 - a 2-[(N-1-(4-N-methylcarboxamidopyrazolyl)] adenosine derivative) and compound 31 (CVT-3033 - a 2-[(4-(1-N-pentyl-pyrazolyl)] adenosine derivative), were found to be short acting functionally selective coronary vasodilators (CV t_0.5 = 5.2 ± 0.2 and 3.4 ± 0.5 min, respectively - rat isolated heart 50% reversal time) with good potency (EC₅₀s = 6.4 ± 1.2 nM and 67.9 ± 16.7 nM, respectively), but they possess low affinity for the ADO A_2A receptor (K_i = 1122 ± 323 nM and 2138 ± 952 nM, respectively; pig striatum).     

A PROCEDURE FOR THE MEASUREMENT OF HEMICHOLINIUM-3 IN BRAIN TISSUE

Abstract— The natural fluorescence of hemicholinium-3 (HC-3) was used to provide a method for determining its concentration in brain tissue following intraventricular administration of the compound to rats and rabbits. When hemicholinium-3 was perfused through the cerebral ventricles of the rabbit, the highest concentration was found in the pons-medulla oblongata. The effect of a number of compounds on the uptake of hemicholinium-3 in the brain was studied in rats. Choline and tetramethylammonium significantly reduced the uptake of HC-3 from the cerebral ventricles but tetraethylammonium, physostigmine and atropine had no effect. The results are discussed in relation to the known effects of hemicholinium-3 in the central nervous system.     

AN IMPROVED CELL FRACTIONATION PROCEDURE FOR THE PREPARATION OF RAT LIVER MEMBRANE-BOUND RIBOSOMES

A cell fractionation procedure is described which allows the preparation from rat liver of a rough microsome population containing almost 50% of the membrane-bound ribosomes of the tissue. The fraction is not contaminated with free ribosomes or smooth microsomes, and, by various other criteria, is suitable for studies of ribosome-membrane interaction.     

純細胞色素C的簡易製備方法及其若干性質

(一)用陽離子交换劑(synthetic zeolite)直接吸附高等動物心肌抽提液一次,並用3.84M硫酸銨作洗脫劑,即可製得含鐵量0.43%的細胞色素c。因此提供了一個大量製備純細胞色素c的簡單方法。 (二)含鐵量0.43%的細胞色素c,它的550mμ和278mμ光密度的比值,視產品的還原程度而定,其範圍從1.13到1.26,我們所製得的產品,其比值在1.23左右。 (三)我們测量了氧化及還原的細胞色素c(含鐵0.43%)從230mμ到600mμ的吸收光譜,並發現和前人所報告的略有不同。氧化細胞色素c在550mμ的消光係數為0.80×10~4,此值與文獻上的數值相差很多。 (四)我們比較了含鐵量0.43%的細胞色素c和含鐵量0.34%的細胞色素c的一些酶性質,證明他們是相同的;並且兩者都可以變成“內源”細胞色素c。 (五)我們認為現有的實驗證據不足以說明純細胞色素c的含鐵量大於0.43%。     

急性重复缺氧对小鼠脑组织腺苷及其A1受体的影响

分别应用酶鉴别分光光度法和放射性配体结合法测定小鼠脑组织腺苷（ａｄｅｎｏｓｉｎｅ,ＡＤＯ）含量及Ａ１受体在急性重复缺氧过程中的变化。发现经急性重复缺氧处理的动物全脑内ＡＤＯ含量有一定程度的累积增加,尤其在海马、脑桥和延髓处的增加较为显著;各脑区Ａ１受体的数目显著低于正常对照组,但海马、脑桥和延髓处Ａ１受体的亲和力显著高于正常对照组。结果提示,重复缺氧后虽然脑内Ａ１受体数目减少,但由于海马、脑桥和延髓处Ａ１受体的亲和力升高,累积增加的ＡＤＯ和Ａ１受体结合后,抑制神经细胞兴奋性的作用仍可能得到加强,从而使ＡＤＯ仍能更好地发挥抑制性神经调制作用。     

A STUDY OF THE ADENOSINE TRIPHOSPHATASE ACTIVITY OF MYOSIN AND ACTOMYOSIN

1. An experimental study was made on the adenosine triphosphatase action of crystalline myosin and actomyosin preparations under different conditions. 2. No enzymatic activity was found in the absence of salts. Activation was given by KCl and CaCl₂, whereas MgCl₂ in the presence of other ions inhibited. 3. The effect of pH is complex. In stabilizing buffers or at low temperature, there are two optima (pH 6.2 to 6.5 and pH 9.2) provided Ca is present. Without Ca only the acid optimum is found. The highest activities are reached in glycine buffer at pH 9.2 in the presence of Ca. 4. The study of the Mg-Ca antagonism revealed that the inhibition due to Mg is fully developed with Mg:Ca ratios less than 1, the inhibition usually exceeding 90 per cent. 5. It is shown that in the muscle the myosin-ATPase is most probably also subjected to the inhibitory action of the Mg ions. 6. From data in the literature it is calculated that the liberation of inorganic phosphate during muscular activity takes place at a rate of at least 0.200 mg. P per mg. myosin per minute. 7. From the results of the present study it is found that the myosin in the muscle can liberate inorganic phosphate from ATP at a rate of at most 0.003 mg. P per mg. myosin per minute. 8. It is concluded therefore that myosin-ATPase cannot be responsible for the liberation of the main part of the phosphate in contracting muscle, and therefore cannot have the rôle in muscular metabolism ascribed to it in recent hypotheses and discussions.     

天然有机合成化学的回顾与展望

本文回顾了八十年代初期以来天然有机合成化学取得的主要进展,并着重从仿生育机合成、生理活性物质的有机合成和立体选择有机合成等方面取得的进展,展望了天然有机合成化学的发展趋势.