Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

Advances in the design of special cryosurgical apparatus in China

Advances in the design of special cryobiomedical apparatus and a review of the trend of developments in the field of cryosurgery in China are discussed. The typical structure of two special cryoprobes for treatment deep in the body and the technology of designing these probes are presented in detail. Some cases which are treated successfully with the above cryoprobes will also be discussed. The experimental aspects of heat transfer in frozen tissue and of the temperature profiles both of a human brain during surgery and of the cryoprobe are described. Other improvements in the field of cryosurgical devices, e.g., four main ways of attaching freezing tips to cryoprobes during surgery and an LN2 transfer tube with high dexterity are also presented. Finally, the development of commercial cryosurgical apparatus in China is also discussed.     

利用人摄金蛋白(METALLOTHIONEIN)启动子和牛乳突瘤病毒在哺乳动物细胞中表达乙型肝炎表面抗原

用人Metallothioenin-Ⅱ启动子、乙型肝炎表面抗原基因,SV40早期基因的编接位点和多聚A位点构成了表达组件,然后插入到经改造过的BpV-1质粒中。所得质粒pdMTsAg-5转染小鼠C127细胞得到转化克隆。Ausria Ⅱ检测证明13株中有12株能产生HBsAg。对其中一株进行HBsAg收率观察,隔天收获为292.6～525.8μg/升,每天收获为200.9～369.0μg/升,可连续收获60天以上。经重金属离子诱导后,收率增至583～854.4μg/升。培养液经超滤浓缩和两次密梯离心后,可集中为一个狭窄的峰,顶峰的Ausria Ⅱ cpm为1.05×10~7,密度为1.20g/ml。     

Phosphorylation of ankyrin decreases its affinity for spectrin tetramer

The effects of phosphorylation on the interaction between spectrin and ankyrin were investigated. Spectrin and ankyrin were phosphorylated using purified human erythrocyte membrane and cytosolic (casein kinase A) kinases. These two kinases have similar properties as well as activities toward spectrin and ankyrin. Both kinases catalyzed the incorporation of about 2 mol of phosphate/mol of spectrin and about 7 mol of phosphate/mol of ankyrin. These phosphates were incorporated primarily into seryl and threonyl residues of the proteins. The phosphopeptide maps of ankyrin phosphorylated by the membrane kinase and casein kinase A were identical. Binding studies indicate that ankyrin exhibits different affinities for spectrin dimers (KD = 2.5 +/- 0.9 X 10(-6) M) and tetramers (KD = 2.7 +/- 0.8 X 10(-7) M). These dissociation constants were not appreciably affected by the phosphorylation of spectrin. On the other hand, phosphorylation of ankyrin was found to significantly reduce its affinity for either phosphorylated or unphosphorylated spectrin tetramers (KD = 1.2 +/- 0.1 X 10(-6) M) but not spectrin dimers (KD = 2.5 +/- 0.4 X 10(-6) M). The same results were obtained using either the membrane kinase or casein kinase A as the phosphorylating enzyme. The above observation suggests that ankyrin phosphorylation may provide an important mechanism for the regulation of the erythrocyte membrane cytoskeletal network.     

元江干热河谷山地五百年来植被变迁探讨

元江河谷是云南省最干热地区之一,在海拔800—900米以下的山地上广泛分布着稀树灌草丛。根据《元江府志》(1714年编纂)、《元江州志》(1826年编纂)、《元江志稿》(1922年编篡)及对现存植被的考察,本文探讨了元江干热河谷山地五百年来植被的变迁。 元江县森林复盖率的减少与人口的增加有密切关系,十七世纪中期以前,森林复盖率在75％以上,十八、十九世纪时为70％左右,1958年为61.5％,1975年为27.3％,至1982年则为19.3％。研究表明,在十九世纪以前,这个地区分布的主要植被是热带季雨林,甚而热带季节雨林,以后热带稀树灌草丛则迅速发展。植被的历史变化与土壤流失密切相关。植物群落的演变是由以乔木树种为优势演变为以灌木种类为优势,再演变为以多年生草木植物为优势,而最后则成为裸地。本文也讨论了这个地区植被恢复的方法。     

蝮蛇毒抗凝血活酶组份及几种蛇毒粗毒对人红细胞和血小板的作用

蝮蛇毒抗凝血活酶组分及蝗蛇毒、圆斑蝰蛇毒和眼镜蛇毒粗毒,不仅能够水解血浆中的磷脂,而且还能水解完整人红细胞膜和完整人血小板膜上的磷脂。但是五步蛇毒和金环蛇毒粗毒却不能水解完整人血小板膜上的磷脂。 扫描电镜观察表明,由于抗凝血活酶组份和几种蛇毒粗毒的作用,人红细胞和人血小板的形态发生了巨大的变化;人红细胞由正常的双圆盘形变成带刺的小球,人血小板的外形变成蜂窝状。     

张士灌区土壤中镉形态的探讨

本文研究了张士灌区土壤中镉的形态。交换态占78％,可给态占18％,不溶态和碱溶态只占3—4％。在田间酸性土壤中,Cl~-是无机配位体的主要成分。镉主要与Cl~-络合而形成[CdCl]~-和CdCl_2。土壤中加入石灰和钙、镁、磷肥、HCO_3~-增多,镉与之生成Cd(HCO_3)_2沉淀。在长期淹水状况下,S~-增多,镉与S~-产生CdS沉淀,难以被水稻吸收。     

长豇豆（Vigna sesquipedalis）生物学特性观察

豇豆近年来公认原产于热带西非(原生中心)和印度次大陆(次生中心)。种子含蛋白质25.0%,在豆类中仅次于大豆,比牛肉、猪肉和鸡肉的蛋白质含量都要高。鲜果含86%的水,3.3%的蛋白质,7.5%的碳水化合物,是广大人民喜爱的蔬菜。我国通常作蔬菜栽培的是长豇豆,又名豆角,其生物学特性的记载并不详尽。为此,我们对长豇豆作了进一步系统的观察,为育种和丰产栽培提供了必要的理论依据。