Molecular cloning of a gene for a member of carcinoembryonic antigen (CEA) gene family; signal peptide and N-terminal domain sequences of nonspecific crossreacting antigen (NCA)

A 2073-base pair DNA fragment containing a part of gene for a member of carcinoembryonic antigen (CEA) gene family, has been isolated from human DNA library after screening with 32P-labeled 53-mer oligodeoxyribonucleotide corresponding to N-terminal 18 amino acids of CEA gene family and cDNA encoding CEA (1,2). The fragment contains two exons; the one encodes the first 60% of signal peptide and the other the rest of it in addition to 107 amino acids which correspond to the N-terminal domain of CEA (1,2). Apparently, the second intron is inserted between the first and the second nucleotides of the codon for 108th amino acid. The presence of Ala instead of Val as the 21st amino acid of the N-terminal domain indicates that the exon encodes nonspecific crossreacting antigen (NCA).     

Immunocytochemical localization of lactoferrin in human neutrophils

Summary The subcellular localization of lactoferrin in human neutrophils was studied by an electron-microscopic immunoperoxidase method. This molecule was detected in small granules of blood polymorphonuclear leukocytes. A morphometrical analysis showed that there was no significant difference in the mean size between lactoferrin-positive and myeloperoxidase-negative granules. In contrast, the mean size of myeloperoxidase-positive granules was significantly larger than that of lactoferrin-positive granules. This indicates that lactoferrin is contained in the myeloperoxidase-negative, secondary, granules of human neutrophils. In immature bone marrow mononuclear neutrophils, lactoferrin was present in cytoplasmic granules of somewhat larger size than lactoferrin-positive granules of polymorphonuclear leucocytes. A morphometrical study showed that the mean size of lactoferrin-positive granules was significantly greater in immature bone marrow cells than in polymorphonuclear leucocytes. This indicates that lactoferrin-positive granules decrease in size as the cells mature. Besides cytoplasmic granules, lactoferrin was demonstrated in the Golgi complex and a part of the rough endoplasmic reticulum of immature bone marrow neutrophils, probably myelocytes and early metamyelocytes. These results show that lactoferrin is synthesized and packed into secondary granules in immature bone marrow neutrophils and therefore that the secondary granules are a type of secretory granule.     

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system

Summary The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobueet al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra-and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin.The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.     

A monoclonal antibody to the carbohydrate chain on human hepatocellular carcinoma-associated antigen which suppressed tumor growth in nude mice

Summary There have been few reports stating that monoclonal antibody alone inhibits human solid tumor growth in vivo. The present study demonstrated that monoclonal antibody S1 (IgG2a), which recognized the antigenic determinant of the carbohydrate moiety, showed antibody-dependent cell (or macrophage)-mediated cytotoxicity (ADCC or ADMC) in conjunction with murine splenocytes of both BALB/c and athymic mice. In vivo experiments demonstrated that the antibody S1 clearly prolonged the survival of athymic mice which had been inoculated with a human liver carcinoma cell line. In addition, the antibody S1 significantly suppressed the human hepatoma line transplanted s.c. into nude mice. ¹²⁵I-Labeled monoclonal antibody S1 revealed that the antibody accumulated significantly in the tumor mass. Many mononuclear cells were observed surrounding tumor cells when the antibody was given. This model system might be useful for analyzing the ADCC (or ADMC) mechanism in vivo.     

Formation of Ascorbate Oxidase in Cultured Pumpkin Cells

Ascorbate oxidase activity rapidly increased during callus formationfrom pumpkin fruit tissue. The activity reached a maximum at5 days after transfer and then declined. In callus which hadbeen subcultured at about 4-week intervals for more than oneyear, the activity also increased after transfer to fresh mediumand reached a maximum in the early logarithmic phase of growth.Light had little effect on the appearance of ascorbate oxidaseactivity in pumpkin callus. In the callus grown in the presenceof 10µM CuSO₄, the activity was about 10 times that inthe presence of 0.1 µM CuSO₄, suggesting that the formatonof ascorbate oxidase in pumpkin callus is stimulated by copper,a prosthetic metal of the enzyme. From 45 to 75% of the totalascorbate oxidase activity in pumpkin cell suspension cultureswas found in the medium. Ascorbate oxidase activity in the medium,as well as that in the cells, increased soon after transferto fresh medium, and reached a maximum at about 5 days. (Received July 2, 1987; Accepted November 21, 1987)     

三个与agropine合成相关的基因在大肠杆菌微细胞中的表达

pTiAch 5 TR区与在植物中积累agropine相关的三个基因片段已导入pINIIA或pACYC184,在大肠杆菌微细胞中表达出杂合蛋白。三个基因片段的pINIIA重组质粒的表达得到稳定蛋白、不稳定蛋白和在两个相表达出相似蛋白的三种结果。基因2'HindⅢ片段上的UGAA序列是在两个相表达相似蛋白的原因。从产生的杂合蛋白分子量和读码方式看,pTiAch 5中基因2'和基因1'的结构与pTi 15955近似,但由基因0'片段产生的杂合蛋白分子量比根据pTi15955 DNA序列数据推测的稍大。从表达强度和微细胞操作看,这个系统还不适应从大肠杆菌中方便地纯化杂合蛋白的需要。     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.