Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

The marginal regions of thylakoid membranes: A partial characterization by polyoxyethylene sorbitan monolaurate (Tween 20) solubilization of spinach thylakoids

The detergent Tween-20 solubilized preferentially portions of the marginal regions of Spinacea oleracea L. thylakoid membranes and, thus, opened the inside of the grana to the external media. Differential centrifugation. following Tween-20 solubilization. enabled separate fractions of grana and stromal-exposed membranes to be isolated. Analysis of Tween-20 solubilized material, after pelleting all membrane material by centrifugation at 100 000 g, revealed polypeptides associated with the coupling factor (CF₁) particles, cytochrome b₆/f and photosystem II complexes, suggesting that the marginal membranes contain these proteins. Concomitantly, the 100 000 g pellet was depleted in cytochrome b₆/f and P700, determined spectroscopically, Thus. our results reveal the margin to be a distinct membrane region, which does not contain the light-harvesting centers of photosystem II (LHC II). The implication of these results, in terms of the energetic interaction of components of granal and stromalexposed membrane regions, is discussed.     

The identification and metabolism of GA9 and its metabolites in seed coats and shoots of pea,line G2

[³H]gibberellin A₉ was applied to shoots or seed parts of G2 pea to produce radiolabeled metabolites. These were used as markers during purification for the recovery of endogenous GA₉ and its naturally occurring metabolites. GA₉ and its metabolites were purified by HPLC, derivatized and examined by GC-MS. Endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ were identified in pea shoots and seed coats. GA₅₁-catabolite and GA₂₉-catabolite were also detected in seed coats. GA₇₀ was detected in seed coats following the application of 1 g of GA₉. Applied [³H]GA₉ was metabolized through both the 13-hydroxylation and 2-hydroxylation pathways. Labeled metabolites were tentatively identified on the basis of co-chromatography on HPLC with endogenous compounds identified by GC-MS. In shoots [³H]GA₅₁ and [³H]GA₅₁-catabolite were the predominant metabolites after 6 hrs, but by 24 hrs there was little of these metabolites remaining, while [³H]GA₂₉-catabolite and an unidentified metabolite predominated. In seed coats [³H]GA₅₁ was the initial product, later followed by [³H]GA₅₁-catabolite and an unidentified metabolite (different from that in shoots), with lesser amounts of [³H]GA₂₀, [³H]GA₂₉ and [³H]GA₂₉-catabolite. [³H]GA₇₀ was a very minor product in both cases. [³H]GA₉ was not metabolized by pea cotyledons.Edited by T.J. Gianfagna.Author for correspondence     

3α, 11α-Dihydroxy-23-oxo-lup-20(29)-en-28-oic acid from Acanthopanax trifoliatus

The new triterpene 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid was isolated from Acanthopanax trifoliatus. Its structure has been determined on the basis of spectroscopic data and chemical transformations.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

外源蜕皮激素对蓖麻蚕蛹发育的效应

本文报道蓖麻蚕蛹在室温28℃下的卵巢发育过程,以及外源20-羟基蜕皮酮对蚕蛹发育的影响。正常蛹在任何发育期内注射20-羟基蜕皮酮后,全部仍羽化成蛾,但蛹期延长约1至4天。无脑蛹经注射后出现蛹——蛾的变态,发育情况因剂量而不同:注射0.1微克后约有半数蛹发育成蛾;注射2微克羽化率较高,卵巢管的发育也最好;4微克或更高的注射量能使全部蛹发育成蛾,但卵巢管多少有些不正常。注射量超过5微克时,蛾体较小,颜色浅黄,没有或只有很少的鳞片。蛹的发育天数随剂量的增大而减少。经外源20-羟基蜕皮酮处理后,无论是有脑蛾或是无脑蛾的卵粒都明显地比正常蛾的卵粒大。当超过一定的注射量时,注射量越大,蚕蛾的自动蜕壳能力越差。     

棉株根系伤流中的细胞分裂素类物质

棉株根系伤流有明显昼夜节奏,白天多,夜晚较少。白天中又以上午9～12时的伤流量为最高。伤流中CTK量及其浓度也以白天为高。在切除地上部4天以后,从蕾期和铃期棉株已不再能收集到伤流液,但盛花期棉株在切除地上部6天以后,仍产生了相当多的伤流,其中仍含有丰富的CTK类物质。盛花期棉株根系伤流量及其中CTK水平高于铃期棉株,提示铃期棉株根系活力已开始趋向衰老。根据Sephadex LH-20柱层析及高效液相层析鉴定出棉株根系伤流中的CTK类物质有Z,ZR,和IPA。     

Ovarian and haemolymph titres of ecdysteroid during the gonadotrophic cycle in Diploptera punctata

The free (non-conjugated) ecdysteroid in the ovaries during the first gonadotrophic cycle of Diploptera punctata was identified as 20-hydroxyecdysone. The hormone, quantified by radioimmunoassay and by ultraviolet absorbance, was detectable in the ovary toward the end of vitellogenesis; the quantity increased rapidly during chorion formation. Ovaries with chorionated eggs contained 67 μg of 20-hydroxyecdysone per g fresh weight. The haemolymph free-ecdysteroid, not identified physicochemically, was quantified by radioimmunoassays. The highest concentration was observed at adult emergence; the titre declined between days 1–3 and then remained at a relatively constant level through oviposition (which occurs between day 7 and 8); titres in pregnant females were higher. Ovariectomized females exhibited the same pattern of ecdysteroid titres in the haemolymph as the sham operated controls throughout the period corresponding to the first gonadotrophic cycle. Thus the ovary may not be the only source of haemolymph ecdysteroid related to reproduction in adult females.     

Monitoring ovulation and implantation in the cynomolgus macaque (Macaca fascicularis) through evaluations of urinary estrone conjugates and progesterone metabolites: a technique for the routine evaluation of reproductive parameters

Investigations were undertaken to determine the applicability of recently reported specific radioimmunoassays for urinary estrone conjugates and progesterone metabolites for monitoring ovarian function in the cynomolgus macaque (Macaca fasciularis) and other macaque species. Mean estrone conjugate measurements appear to accurately reflect the preovulatory estrogen peak in both conceptive (n = 5) and nonconceptive (n = 6) cycles, as well as to indicate early pregnancy through increases which are significantly elevated by Day + 15 (p less than 0.049) post estrone conjugates peak. The mean luteal phase levels of these progesterone metabolites are significantly elevated by Day + 14 (p less than 0.012) in conceptive cycles when compared to the mean values for nonconceptive cycles.