Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

壳聚糖固定化木瓜蛋白酶的研究

以壳聚糖为载体,成二醛为交联剂将木瓜蛋白酶固定化。5％戊二醛在4-6℃下处理载体5h,加酶液(3.5mg/mL蛋白,pH7.2)固定12h,活力回收达32％,作用于酪蛋白的半衰期为36天,其表观K_m(酪蛋白)值为0.075％(W/V),溶液酶的K_m值为0.086％;最适pH7.0～7.5,溶液酶为7.0～8.5。固定化酶在pH8.5以下,溶液酶在9.0以下活力稳定。固定化酶在45℃以下,溶液酶在75℃以下稳定。用6mol/L脲洗脱固定化酶4次(5.5h)活力仍有54.5％。用固定化酶处理啤酒浊度比对照下降了1.5-3.7倍,蛋白质含量下降了44％,冷藏(4℃)120天无冷混浊现象发生并保持了啤酒原有风味和理化性状。     

转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用~(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。     

Purification and characterization of a pp60c-src-related tyrosine kinase that effectively phosphorylates a synthetic peptide derived from p34cdc2.

A protein tyrosine kinase has been purified from the particulate fraction of bovine spleen to a specific activity of 0.217 mumol/min/mg at 100 microM ATP and 3 mM [Val5] angiotensin II. Both the angiotensin phosphorylation activity and immunoreactivity towards an antibody preparation raised against a synthetic peptide containing the autophosphorylation site of pp60c-src, Cys-src(403-421), were monitored during the purification. The purified sample displayed three closely spaced protein bands with molecular weights of 50-55 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All bands could be phosphorylated exclusively on tyrosine residues under autophosphorylation conditions. All reacted on immunoblots with an antibody raised against a synthetic peptide corresponding to the consensus autophosphorylation site of members of the pp60c-src family of tyrosine kinases. Tryptic phosphopeptide maps of the three proteins were essentially indistinguishable. The results suggest that the purified enzyme preparation contained mainly three closely related pp60c-src-family protein tyrosine kinases or a pp60src-family protein tyrosine kinase modified posttranslationally to give three closely spaced protein bands on sodium dodecyl sulfate gel. Neither of these proteins appears to be pp60c-src or p56lck. The spleen protein tyrosine kinase was found to phosphorylate a p34cdc2 kinase peptide, Cys-cdc2(8-20), which contained the regulatory tyrosine residue Tyr-15 about 20 times better than [Val5]angiotensin II or Cys-src(403-421) peptide at a peptide substrate concentration of 1 mM. In contrast, epidermal growth factor receptor kinase partially purified from A431 cells did not show preference for Cys-cdc2(8-20) as its substrate. Although Cys-cdc2(8-20) contained two tyrosine residues, only the tyrosine corresponding to Tyr-15 in p34cdc2 was phosphorylated by the spleen tyrosine kinase. The observation suggests that the primary structure surrounding Tyr-15 of p34cdc2 contains substrate structural determinants specific for the spleen tyrosine kinase.     

Fast sampling,rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Summary Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 l) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na⁺-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.     

Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi.     

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.     

四川省自贡市幼儿园3至5岁儿童乙型肝炎病毒感染情况及影响因素

为了解四川省自贡地区3～5岁幼儿乙型肝炎病毒(HBV)感染情况,并探索与感染有关的因素,1985年调查了1167名幼儿,其HBV总感染率为41.13％,HBsAg阳性率12.68％。幼儿的HBV感染与母亲HBsAg阳性密切相关。共检查母亲409例,38例HBsAg阳性,其幼儿HBsAg阳性率为50％(19/38),HBsAg阴性的母亲371例,其幼儿HBsAg阳性率9.97％(37/371),来自HBsAg阳性母亲的阳性子女占33.3％(19/56)。1986年随访HBV易感幼儿448例,HBV年感染率为12.95％(58/448),HBsAg年阳转率3.79％。HBV年感染率与原幼儿班级HBsAg阳性率的高低有关。     

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.