Biosynthesis of two stage-specific membrane proteins during transformation of Plasmodium gallinaceum zygotes into ookinetes

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.     

Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.     

Characterization of major histocompatibility antigens on trinitrophenyl-modified cells.

Cell membrane proteins encoded for by the major histocompatibility complex (MHC)¹ are associated with the antigenic determinant(s) recognized on trinitrophenyl (TNP)-modified cells by syngeneic murine cytotoxic T lymphocytes and by hapten-reactive guinea pig T cells. To investigate the relationship of the TNP moiety on TNP-modified cells to these major histocompatibility antigens, peritoneal exudate cells or splenocytes from two inbred guinea pig strains and one inbred murine strain were TNP-modified, radioiodinated and lysed in detergent. TNP-derivatized proteins were then isolated using an anti-TNP immunoabsorbent, and the presence on TNP-derivatized histocompatibility antigens in the eluted proteins was determined by immunoprecipitation experiments and SDS-polyacrylamide gel electrophoretic analysis. Whereas most of the various histocompatibility antigens examined were found to be TNP-derivatized in amounts proportional to the degree of membrane protein derivation as a whole, only small amounts of TNP-modified strain 2 guinea pig Ia antigens were found, and no hapten-modified strain 13 guinea pig Ia antigens were detected. It is concluded that, in contrast to most MHC gene products, strain 13 Ia antigens are not derivatized on TNP-modified cells and, thus, represent an important exception demonstrating that histocompatibility antigens need not be directly TNP-derivatized for T cell recognition and activation.     

Multiplicity of allergens in peanuts

Crude peanut protein fractions from raw and roasted peanuts were examined in the RAST with 10 sera from patients showing clinical peanut sensitivity. The radioactive uptake results, which were generally high, did not reveal any distinguishable pattern. Two commercially available peanut proteins, peanut lectin and phospholipase D, gave poor RAST responses. Three purified peanut proteins, α-arachin, conarachin I, and concanavalin A-reactive glycoprotein, all gave significant RAST results that were generally lower than those obtained with the crude extracts. The extent of RAST inhibition obtained with these materials was inversely related to their abundance in the total peanut protein. Crossed immunoelectrophoresis with extracts from raw and roasted peanut indicated the presence of 22 and 10 anodically migrating antigens, respectively. Sixteen IgE binding antigens were revealed for raw peanut and seven for roasted peanut after incubation with a mixed serum from the 10 patients in crossed radioimmunoelectrophoresis (CRIE) using ¹²⁵I-labeled anti-IgE. CRIE plates treated with individual serum samples showed that all the patients had specific IgE for the major antigen peak, which has been tentatively identified as α-arachin. This major storage protein of peanut, which is known to be particularly heat resistant, may be of greater clinical significance than its apparently low RAST activity would seem to indicate.     

毛细管区带电泳测定尿17-羟皮质类固醇

目的 :应用高效毛细管区带电泳 (CZE)技术分离和检测人尿内 1 7-羟类皮质类固醇 (1 7-OHCS) ,以解决微色谱柱 -分光光度测定尿 1 7-OHCS时 ,多酚类化合物对测定的干扰。方法 :标本用活性高岭土吸附尿内色素 ,以茶碱作内标 ,中性苯乙烯 -二乙烯苯树脂微色谱柱吸附尿中 1 7-OHCS。乙醇解吸附 ,采用毛细管区带电泳进一步分离并测定。结果 :运用CZE法分离了人尿 1 7-OHCS ,皮质醇浓度在 2～ 5 0mg .L- 1 范围内具有良好的线性关系 (r=0 .994) ,方法平均回收率 96 .1 7% ,日内及日间CV均小于 5 % ,最低检测限度为 0 .3mg .L- 1 。结论 :与微色谱柱 -分光光度法相比 ,CZE法检测尿内 1 7-OHCS无干扰 ,方法简便、快速、可靠。     

Methods of detecting mature human Ia-like antigen and their metabolic precursors

Ia antigens were shown to be present in the cell almost exclusively as mature alpha beta dimers which split into separate alpha and beta chains after boiling in SDS. In contrast metabolically labelling the cells with [35S]methionine resulted in only free alpha and beta chains being labelled. It is concluded that this widely used type of labelling, although useful for studying intermediate synthesis, should not be used for labelling mature cell surface molecules.     

大鼠膈腹膜间皮孔的电镜观察

用扫描电镜和透射电镜观察了正常大鼠膈腹膜间皮，并观察了腹膜腔内注射中国墨汁和兔血液后大鼠膈腹膜间皮的变化以及腹膜腔和间皮下毛细淋巴管的关系。     

Electrophoretic investigation of nuclear membrane ribonucleases of rat liver and hepatoma-27 cells

Preparations of the nuclear membranes were obtained from purified nuclei of rat liver and hepatoma-27 cells, and from them enzyme-containing extracts of acid-soluble proteins were then prepared. The protein extracts were subjected to disk electrophoresis in 15% polyacrylamide gel. Ribonucleases (RNases) which are constituents of the acid-soluble proteins of the nuclear membranes of normal liver were found to be present as several different components which differed in their electrophoretic mobility and in several physicochemical properties from crystalline bovine RNase and the RNase of nuclear chromatin.Institute of Problems in Oncology, Academy of Sciences of the Ukrainian SSR, Kiev. N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR R. E. Kavetskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 670–672, December, 1977.     

应用激光电泳技术对MA737小鼠乳腺癌细胞膜表面电荷特性的研究

本工作应用激光多普勒电泳技术对ＭＡ７３７小鼠乳腺癌细胞膜表面电荷特性及高温（４２．５℃）对癌细胞的热效应进行了深入的研究。结果发现，ＭＡ７３７癌细胞的电泳迁移率（ＥＰＭ）明显高于相应正常细胞的ＥＰＭ（约２０％），表明癌细胞表面负电荷增高；实验发现唾液酸可能是ＭＡ７３７癌细胞膜表主要负电基团的来源之一。研究还发现，ＭＡ７３７癌细胞的热敏性比相应的正常细胞强，随高温受热时间的延长，癌细胞的ＥＰＭ呈下降趋势。     

Human myeloma light chains with increased molecular weight: high frequency among lambda chains

The discovery of a human myeloma protein comprising a kappa L-chain with an increased mol. wt of 30,000) (Bouvet et. al., 1980) prompted investigations on the incidence of such heavier L-chains among other human myeloma proteins. In 105 samples examined, 34 were found to have L-chains heavier than normal (23,000-24,000), ranging from 25,000 up to 31,000, and five of lighter mol. wt (21,000-22,000). These mol. wt abnormalities were detected by electrophoresis in sodium dodecyl sulfate 10% polyacrylamide gels (SDS-PAGE) after reduction with 2-mercaptoethanol. The mol. wt of three of the heavier kappa or lambda chains was also estimated by filtration through a Sephadex G100 column and by sedimentation equilibrium. All three methods indicated a mol. wt increase of about 15-25% as compared with the usual mol. wt. The distribution of the high mol. wt chains among all L-chains examined was found to be 11 out of 62 kappa chains (17.7%) and 23 out of 43 lambda chains (53%) (P less than 0.001). A preferential association of such L-chains with H-chains producing multiple bands in SDS-PAGE (P less than 0.01) and an association between multiple L-chain and multiple H-chain band (P less than 0.05) were also observed. In contrast, no abnormal L-chain was found in immunoglobulins from normal subjects. Spontaneous degradation of the normal H-chains sometimes yielded fragments of 30,000 mol. wt. These fragments were easily distinguishable from abnormal L-chains. The nature of extra mol. wt in heavy L-chains was investigated for the presence of carbohydrate moiety. Four large and three normal size L-chains were examined for amino-sugar and sialic acid content. A small amount (one residue per molecule) of amino-sugar was detected only in two normal and two heavy L-chains, whereas sialic acid was only found in the heaviest (27,000-30,000) L-chains (Lh) and in small percentage (one or two residues per molecule). Total sugar estimation in one Lh chain indicated a proportion not exceeding three or four residues per L-chain (mol. wt 1,000) and this is insufficient to explain the 15-25% (3,600-6,000) mol. wt increase. It is therefore possible that, at least in some heavy myeloma L-chains, an additional peptide is expressed. Whatever the nature of the increase it would be of interest to elucidate whether this is a marker of malignant process or of an intermediate step of normal Ig synthesis.