POSTSYNAPTIC ADRENERGIC-CYCLIC AMP CONTROL OF THE SEROTONIN CONTENT OF CULTURED RAT PINEAL GLANDS

Abstract— This investigation was designed to determine whether the amount of serotonin (5-HT) in cultured pineal glands can be altered by norepinephrine (NE). Treatment with l -NE (10^?5-10^?7m ) for 4-6 h caused a gradual decrease in the concentration of 5-HT to a value that was less than 30% of that in the untreated control gland. This effect was observed using chronically denervated pineal glands. d -Norepinephrine (10^?6-10^?7m ) and dopamine (10^?4m ) were ineffective in lowering 5-HT. The effect of l -NE was completely blocked by a β-adrenergic receptor blocker, propranolol and was only slightly decreased by α-adrenergic receptor blockers. These observations indicate that l -NE acts post-synaptically via a highly specific β-adrenergic mechanism. The effect of l -norepinephrine was mimicked by theophylline and N⁶, 2′0-dibutyryl adenosine 3′,5′-monophosphate, an indication that adenosine 3′,5′-monophosphate is involved in the effect of l -NE on 5-HT. Treatment with cycloheximide, which by itself caused a decrease in pineal 5-HT, also blocked any further decrease caused by treatment with l -NE, an indication that protein synthesis is necessary for maintenance of baseline levels of serotonin and for the effect of l -NE to be observed. The total amount of l -[³H]NE and degradation products of L-[³H]NE in the gland after 6 h of treatment with l -[³H]NE was less than 3 pmol. This amount of l -NE and degradation products of l -NE could not account for the decrease of 100-200 pmol of 5-HT on the basis of a mole for mole replacement of 5-HT by l -NE. These findings are consistent with the hypothesis that non-neuronal pineal 5-HT is physiologically regulated by the release of l -NE from the sympathetic nerve network.     

LIPID COMPOSITION OF HUMAN, BOVINE AND SHEEP PINEAL GLANDS

Abstract— —The lipid composition of human, bovine and sheep pineal glands was determined. No characteristic species difference was found in lipid content and composition. The total lipid was 2·9–4·0 per cent of wet weight of which phospholipid comprised 58–71 per cent and cholesterol 13·9–15·8 per cent in the three species. The phospholipid composition was 45 per cent phosphatidyl choline, 22 per cent phosphatidyl ethanolamine, 14 per cent sphingomyelin, 9 per cent phosphatidyl serine, 8 per cent monophosphoinositide, and 2 per cent diphosphatidyl glycerol. The major fatty acids found were C14:0, C16:0, C18:0, C16:1, and C18:1. In contrast to other tissues, pineal sphingomyelin has a low C24:1 content. No significant amounts of polyphosphoinositides or gangliosides were detected. When its lipid composition is compared with that of a number of other tissues, pineal is found to be most similar to testes and unlike pituitary and brain.     

DIURNAL RHYTHM AND TURNOVER OF TRYPTOPHAN HYDROXYLASE IN THE PINEAL GLAND OF THE RAT

Tryptophan hydroxylase in the pineal gland of the rat was found to undergo a diurnal rhythm in activity with an elevated activity at night. The rhythm was abolished in constant light. Cycloheximide (15 mg/kg, i.p.), administered both at night and during the day, caused a rapid decay in activity suggesting that tryptophan hydroxylase was subject to a rapid turnover in vivo. The primary site of control appeared to be at the level of translation since actinomycin D had no effect. Some relevant properties of the enzyme were studied. Thiol-containing compounds were shown to substantially protect pineal tryptophan hydroxylase from inactivation at 0°C but provided little protection at higher temperatures. The inactivation process appeared to be independent of oxygen. The activity of the enzyme, lost after ageing at 0°C. could be recovered by incubation with dithiothreitol under anaerobic conditions. Fresh enzyme, or enzyme inactivated at 37°C could not be activated by this process. A re-examination of the action of p-chlorophenylalanine (PCPA) on pineal tryptophan hydroxylase revealed that an irreversible inactivation occurred within 6h (25% of initial activity) followed by a recovery within 24 h. The rapid turnover of the enzyme is the probable reason for the failure of previous studies to observe an irreversible inhibition of this enzyme by PCPA.     

SUBCELLULAR LOCALIZATION OF TRYPTOPHAN-5-MONO-OXYGENASE IN BOVINE PINEAL GLANDS AND RAPHE NUCLEI

Abstract— Tryptophan-5-mono-oxygenase from both bovine raphe nuclei and pineal glands was activated by preincubation with dithiothreitol and ferrous ion at pH 8.5. The optimum pH for the enzyme activity lies between pH 6.4 and 7.3. Preincubation increased the activity of the enzyme from raphe nuclei by about 20 times in both the homogenate and 105,000 g precipitate prepared from it. Activity in the 105,000 g supernatant fraction was about trebled. Corresponding increases in pineal gland enzyme activity were noted: 100 times in homogenate and 105,000 g precipitate and 15 times in 105,000 g supernatant fluid. Total recoveries of activated enzyme from the homogenate prepared in hypo-osmotic medium, in the 105,000 g supernatant and precipitate, were 87.1% and 79.0% for raphe nuclei and pineal glands respectively. Of this, 89.5-91.3% in the case of the raphe nuclei and 76.0-82.0% in the case of the pineal glands, was found in the precipitate. In contrast, 85-90% of the lactate dehydrogenase activity was found in the supernatant fraction. The results of subcellular fractionation revealed that the raphe nuclear enzyme was located in both 'mitochondrial' and 'microsomal' fraction while the pineal gland enzyme was effectively restricted to the 'mitochondrial' fraction. The structural characteristics of the fraction were confirmed by electron microscopy.     

AN EXAMINATION OF THE CATALYTIC PROPERTIES OF SEVERAL AMINOTRANSFERASES IN BRAIN AND LIVER BY MEANS OF AN IMPROVED RADIOMETRIC ASSAY WITH DINITROPHENYLHYDRAZINE

Abstract— The assay of aminotransferases, performed by solvent extraction of keto acids formed from labelled amino acids, has been modified to enhance the recovery of both aliphatic and aromatic keto acid products. The keto acids are first converted to their respective dinitrophenylhydrazones which are more completely extracted into less polar organic solvents. By this manoeuvre, both keto acid extraction is increased and the extraction of the precursor amino acid is reduced. Employing this technique, the kinetics of brain-stem γ-aminobutyric acid (GABA), tryptophan, 3,4-dihydroxyphenylalanine (DOPA) aminotransferases and brain-stem and liver tyrosine aminotransferases were examined. Brain-stem aminotransferases, particularly the aromatic amino acid transferases, have a higher affinity for both the amino acid and the keto acid when the aromatic keto acid, phenylpyruvate (0·8 mM), is employed as amino group acceptor, whereas maximal velocities for aminotransferase reactions are much greater when α-ketoglutarate (0·8 m m ) is the amino group acceptor. Brain-stem tyrosine aminotransferase exhibits a much lower affinity for tyrosine in the presence of either 0·8m m -α-ketoglutarate or 0·8 m m -phenylpyruvate than does liver tyrosine aminotransferase. p -Chlorophenylpyruvate and phenylpyruvate exhibit similar properties as amino group acceptors for brain-stem tryptophan aminotransferase. Cysteine inhibits tryptophan aminotransferase when phenylpyruvate is the amino group acceptor, in a manner which is competitive with the amino acid. Benzoylformate inhibits both tryptophan and DOPA aminotransferases when phenylpyruvate is the amino group acceptor, but this inhibition does not appear to be competitive with phenylpyruvate.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

INCREASE OF BRAIN TRYPTOPHAN BY ELECTROCONVULSIVE SHOCK IN RATS

—ECS markedly increased tryptophan and 5-hydroxyindoleacetic acid levels in brain. Brain serotonin and plasma tryptophan levels were unaffected.     

STIMULATION OF SEROTONIN N-ACETYLTRANSFERASE IN PINEAL ORGAN CULTURE BY DRUGS

Abstract— Drugs such as cocaine, procaine, pheniprazine (Catron) and veratridine, which have actions on sympathetic nerves and nerve terminals, were examined for their ability to increase serotonin N-acetyltransferase (EC 2.3.1.5; NAT) in pineal organ culture. The absence of potassium (0 KCl) was also examined. NAT is known to respond to β-adrenergic stimulation. It was found that these drugs and 0 KCl increased the enzyme activity 100 to 2000-fold in innervated pineals but had virtually no effect in denervated pineals. The effects on innervated pineals were blocked by the β-blocker propranolol but not by the α-blocker, phentolamine. These drugs and 0 KCl inhibited to varying degrees [³H] 1-norepinephrine uptake in pineals. It is concluded that these agents activated the β-adrenergic receptor on pineal cells by causing an accumulation of extraneuronal norepinephrine. The accumulation of norepinephrine is due, at least in part, to the blockade of norepinephrine reuptake by nerve terminals. The ability of veratridine to stimulate NAT and to inhibit norepinephrine uptake was reversed by tetrodotoxin, a blocker of sodium permeability in excitable tissue, thus veratridine acts by increasing sodium permeability in nerve terminals. This adds support to the theory that catecholamine uptake is a process that requires a sodium gradient across the nerve terminal membrane.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

应用免疫荧光技术检测肺微小根毛霉的感染

近几年因医源性导致患者机体免疫功能低下或受损有所增加,从而使深部丝状真菌感染有逐年上升的趋势,由于检测上的种种原因,大部份是尸检后发现,难以鉴定到属种,对临床治疗带来直接影响,为进一步探讨丝状真菌的快速诊断,我们从肺活组织中分离出的微小根毛霉,注射接种到不同剂量的~(60)Co-γ辐照鼠体内后,间隔一定时间,用间接免疫荧光法检测,发现3GY辐照后14—28日检出率是41.87%(皮下注射),66.66%(腹腔注射),7GY、5GY、1GY的辐照均低于3GY,除与根霉有交叉反应外,曲霉、青霉、白色假丝酵母等均无交叉反应。     

抗癌药物紫杉醇的全合成—Holton合成紫杉醇路线的剖析

从全合成战略与路线,关键反应,保护基应用和立体化学方面对Holton的线性全合成紫杉醇路线进行剖析归纳。     

RAT BRAIN STEM TRYPTOPHAN HYDROXYLASE: MECHANISM OF ACTIVATION BY CALCIUM

Abstract— The activity of soluble tryptophan hydroxylase from rat brain stem was increased in presence of mm concentrations of calcium. Similarly to that observed by treating the enzyme with sodium dodecyl sulphate or trypsin, this activation resulted mainly from an increased affinity of tryptophan hydroxylase for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). In addition, the optimal pH for the enzymic activity was shifted from 7.6 to 7.9 following activation by calcium, sodium dodecyl sulphate or trypsin.
Under the assay conditions used for measuring tryptophan hydroxylase activity, calcium also stimulated a neutral proteinase. This latter enzyme could be eliminated from the solution of tryptophan hydroxylase by filtration through Sephadex G 200. The resulting partially purified tryptophan hydroxylase could be activated by calcium only when the neutral proteinase was included in the assay mixture. In support of this conclusion, the effect of calcium on tryptophan hydroxylase was very small in the new born rat when the activity of the neutral proteinase was low. In addition, the activating effect of Ca²⁺ could be antagonized not only by a chelating agent like EGTA but also (partially) by specific inhibitors of proteinases such as benzethonium and PMSF.
These results strongly suggest that the activation of tryptophan hydroxylase by calcium is the consequence of a partial proteolysis of the enzyme by the calcium-dependent neutral proteinase. Therefore, the physiological significance of this irreversible effect is doubtful.     

马桑内酯对培养的大鼠海马星形胶质细胞的激活

实验研究了马桑内酯对纯化培养的大鼠海马星形胶质细胞的影响。发现：（1）免疫细胞化学染色证实马桑内酯作用2h可引起胶质细胞核内NF-кBp65的表达,8h达高峰,至24hNF-кBp65阳性胞核的百分率和平均光密度仍维持在高水平;预先用PDTC作用1h可完全抑制NF-кBp65的核表达,而TNFα单抗则可延迟NF-кBp65免疫反应阳性胞核出现的时间,阳性胞核百分率及其平均光密度也有所下降;（2）酶联免疫吸附实验发现马桑内酯可促进星形胶质细胞培养基内TNFα含量升高,PDTC可部分对抗此作用,本实验表明马桑内酯激活星形胶质细胞的作用部分是由TNFα介导的。     

时间分辨荧光免疫分析法确定血痕种属

采时时间分辨荧光免疫分析法（ＴＲＦＩＡ）鉴定微量血痕种属。固相和铕标记抗体均采用抗人ＩｇＧ抗体。通过检测可确定血痕是否来源于人体。使用本法检测血清样品的灵敏度达５０万倍稀释度,检测血痕样品达２０万倍稀释度,时间分辨荧光免疫分析法具有无放射性污染,标记物保存期长,特异性好,结果稳定,操作简便等优点,适于法医学的常规检测。     

用改进的ELISA法检测腺病毒抗原

用改进的ELISA法检测94份咽拭子标本中的腺病毒抗原,标本先接种人肾细胞,使病毒有一定程度的增殖,同时与传统的病毒分离作对比,结果表明,腺病毒分离阳性的18例,ELISA法检测全部阳性,76例分离阴性(盲传3代)者ELISA法也全都阴性,两者完全相符,ELISA法检测增殖24、48、72小时的细胞培养物腺病毒抗原的阳性率分别为16.7％,72.2％和83.3％,本法特异、可靠、判断客观、可用来检测腺病毒抗原。