Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Subcellular compartmentation of different lipophilic fluorescein derivatives in maize root epidermal cells

Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM) 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester) - BTB bis-trispropane - CDCF (CDCF-DA) 5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative) - DAPI 4,6-diamidino-2 phenylindole dihydrochloride - DMSO dimethylsulfoxide - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethane-sulfonic acid - SNAFL-1 (SNAFL-1-DA) carboxyl SNAFL-1 (its diacetate) - SNARF-1 (SNARF-1-AM) carboxyl SNARF-1 (its acetoxymethyl acetate)     

黑曲霉糖化酶高产和低产菌株糖化酶基因调控区的克隆及其分析比较

以PCR合成的糖化酶高产菌株黑曲霉(Asp. Niger)T21糖化酶基因5’近端非编码区588bp(EcoRI-BamHI)的序列为探针,从T21染色体DNA中克隆到近2.0kb的糖化酶基因5’端非编码区序列,并以此序列为探针从糖化酶低产菌株黑曲霉3.795(T21的诱变出发株)的染色体DNA中克隆到1.5kb的糖化酶基因5’端非编码区序列。该二序列的分析测定结果表明,其结构特征与文献报道的黑曲霉糖化酶基因5’端非编码区的基本一致,被称为“核心启动子”(Core promoter)的TATAAAT框及GCAAT框,分别在翻译起始点的-109bp及-178bp处。此外,在曲霉amdS,amyB基因中已发现有调控功能的CCAAT序列存在于-449bp和-799bp处。高产和低产菌株糖化酶基因5’端非编码区序列的分析比较结果表明,有9个部位的碱基发生了变化。此实验结果为进一步研究黑曲霉糖化酶基因在转录水平上的调控规律打下了基础。     

水解淀粉的酿酒酵母菌的构建

把黑曲霉糖化酶cDNA,酵母磷酸甘油激酶基因启动子区和终止区以及酵母Ty因子的δ序列构建成整合型的糖化酶表达分泌质粒pKG 1。该质粒转化酿酒酵母Y33得到整合型转化子。转化子分泌糖化酶活力在3.0μ/ml以上,在以5％可溶性淀粉为碳源的培养基中静止培养7d,淀粉利用率达86％,生成酒精的浓度与以5％葡萄糖为碳源时相等。     

Simplification and analysis of models of calcium dynamics based on IP3-sensitive calcium channel kinetics.

We study the models for calcium (Ca) dynamics developed in earlier studies, in each of which the key component is the kinetics of intracellular inositol-1,4,5-trisphosphate-sensitive Ca channels. After rapidly equilibrating steps are eliminated, the channel kinetics in these models are represented by a single differential equation that is linear in the state of the channel. In the reduced kinetic model, the graph of the steady-state fraction of conducting channels as a function of log10(Ca) is a bell-shaped curve. Dynamically, a step increase in inositol-1,4,5-trisphosphate induces an incremental increase in the fraction of conducting channels, whereas a step increase in Ca can either potentiate or inhibit channel activation, depending on the Ca level before and after the increase. The relationships among these models are discussed, and experimental tests to distinguish between them are given. Under certain conditions the models for intracellular calcium dynamics are reduced to the singular perturbed form epsilon dx/d tau = f(x, y, p), dy/d tau = g(x, y, p). Phase-plane analysis is applied to a generic form of these simplified models to show how different types of Ca response, such as excitability, oscillations, and a sustained elevation of Ca, can arise. The generic model can also be used to study frequency encoding of hormonal stimuli, to determine the conditions for stable traveling Ca waves, and to understand the effect of channel properties on the wave speed.     

Overall Informativity,OI, in DNA Polymorphisms Revealed by inter-AluPCR: Detection of Genomic Rearrangements

We studied two systems of multilocus markers revealed by PCR using primers directing amplification betweenAlurepeats in a tail-to-tail orientation. Genomic polymorphisms were detected as the presence or absence of the electrophoretic bands representing DNA fragments of a given length. A total of 104 such fragments segregating as Mendelian markers in a panel of eight CEPH families were analyzed by two-point linkage analysis. Fifty-one of these fragments were localized with respect to CEPH markers; they represented 33 loci, 7 of which were multiallelic. Locus-specific oligonucleotides were developed and used as hybridization probes to identify the mapped loci within a complex pattern of inter-AluPCR products. A great proportion of inter-AluPCR polymorphisms represented length variants within amplified DNA segments, while others were presumably due to mutations within the priming sites. To describe the expected number of informative loci per typing experiment we introduced a parameter called overall informativity (OI), which provides a single measure of the multiplex ratio and the informativity of markers contributing to a multilocus system (OIof a single locus is equivalent to its heterozygosity and cannot exceed 0.5 for a biallelic codominant marker). HighOIvalues (5.8 and 11.5) of the two presented systems of inter-AluPCR markers of random chromosomal distribution render them suitable for mapping genomic rearrangements such as genomic deletions in tumoral tissues. This was illustrated by the detection of loss of heterozygosity in the 9q22–qter region in sporadic colon cancer.     

不同森林群落结构与光能利用率的关系

本文在人工落叶松纯林及人工落叶松与水典柳混交林的林冠观测数据的基础上建立了落叶松松和水曲柳的树冠锥体模型。通过对上述两种森林群落结构的太阳辐射的观测,利用电磁波的吸收,反射和透射理论分别对以上两种森林群落的光能利用率进行了计算。结果表明理论计算值与实测结果基本一致;双层次混交林的光能利用率高于单层纯林的光能利用率。     

江苏野菜资源的利用与开发

江苏野菜资源丰富,共计１９２种,隶属４４科１０８属,其中蕨类植物７科１５属５１种,种子植物３７科９３属１４１种。江苏野菜利用历史悠久,近年已发展成为规模种植,产生良好的经济效益和社会效益。