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).     

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.     

A single amino acid substitution converts cytochrome P450(14DM) to an inactive form, cytochrome P450SG1: complete primary structures deduced from cloned DNAS

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.     

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.     

CT-guided stereotactic aspiration of intracerebral hematoma--result of a hematoma-lysis method using urokinase

CT-guided stereotactic aspiration was performed in the CT room on 97 patients with hypertensive intracerebral hematomas, using a standard ventricular cannula. Residual hematomas were liquefied by urokinase and aspirated through the drainage tube. Major and minor rebleeding were seen in 7 cases. Two out of the 4 major rebleeding cases were followed by craniotomy, while the other cases were treated conservatively. More than 80% of the hematomas were aspirated in 68 cases, 50-70% in 19 cases and 30-40% in 6 cases. Operation in the CT room and hematoma lysis with urokinase is very useful for the aspiration of intracerebral hematomas.     

Expression in Escherichia coli of chemically synthesized gene for the human immune interferon.

A 454 base pair fragment of double stranded DNA consisting of a gene for a human immune interferon (hIFN-gamma), initiation and termination signals plus appropriate restriction endonuclease sites, was totally synthesized. The synthesis involved preparation of 62 oligodeoxyribonucleotides by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. This synthetic gene was expressed in E. coli under the control of the lac UV5 promoter. The product has antiviral activity which was acid labile and completely neutralized by antiserum to hIFN-gamma but not by antiserum to hIFN-alpha or hIFN-beta. Molecular weight of hIFN-gamma produced by E. coli was estimated to be about 32,000 and 17,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively.     

Successful artificial insemination in the giant panda (Ailuropoda melanoleuca) at ueno zoo

Successful breeding of the giant panda (Ailuropoda melanoleuca) following artificial insemination was achieved at the Ueno Zoo in 2 consecutive years (1985 and 1986). The first cub, born in June 1985, unfortunately died 43 hours after birth from being crushed by the mother panda; the second cub, born in June 1986, has been growing in good health. Electroejaculation and artificial insemination procedures were performed after immobilization with diazepam (0.1 mg/kg) and ketamine HCL (4.0–5.0 mg/kg). Semen of the male panda was collected by electroejaculation using a rectal probe with a diameter of 2.0 cm and with eight rings as electrodes. Stimulation of the male was given with 3 V (30–40 mA) over a 5-sec period with 5-sec intervals. The female panda exhibited estrus between late February and early March in 1985 and also between mid-january and early February 1986. Increased excretion of urinary total estrogen showed coincidentally at maximum behavioral estrus, and a gradual rise of pregnanediol level was followed by artificial insemination. The gestational length for the first pregnancy was 110 days and that of the second 121 days.     

Identification of membrane anchoring site of human renal dipeptidase and construction and expression of a cDNA for its secretory form

The chemical properties of human renal dipeptidase (hrDP) purified from the membrane fraction of kidney have been characterized. When treated with phosphatidylinositol-specific phospholipase C, hrDP was released from renal membrane fractions. After digestion with trypsin, carboxyl-terminal peptide was isolated employing anhydrotrypsin-agarose column chromatography and reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide was identified at positions 363-369 in the primary structure deduced from the cDNA sequence (Adachi, H., Tawaragi, Y., Inuzuka, C., Kubota, I., Tsujimoto, M., Nishihara, T., And Nakazato, H. (1990) J. Biol. Chem. 265, 3992-3995). Further examination of the chemical composion of the peptide showed that it contained, respectively, 2, 1, 5, 1, and 1 mol of ethanolamine, glucosamine, mannose, inositol, and phosphate in addition to amino acids. These results suggest that the mature hrDP molecule lacks the carboxyl-terminal hydrophobic peptide extension predicted from the cDNA sequence and is anchored at Ser369 via glycosylphosphatidylinositol to the membrane. To characterize further the action of the enzyme, we have established expression systems for both secretory and membrane anchored forms of hrDP using COS-1 cells and found that both recombinant forms were as active as natural enzyme. Our expression system made it possible to prepare large amounts of soluble enzyme, and will contribute toward elucidation of the physiological roles of the enzyme.     

Interconversion of aflatoxin B1 and aflatoxicol by several fungi

Four fungal strains, namely, Aspergillus niger, Eurotium herbariorum, a Rhizopus sp., and non-aflatoxin (AF)-producing Aspergillus flavus, which could convert AF-B1 to aflatoxicol (AFL), could also reconvert AFL to AF-B1. The interconversion of AF-B1 to AFL and of AFL to AF-B1 was ascertained to occur during proliferation of the fungi. These reactions were distinctly observed in cell-free systems obtained from disrupted mycelia of A. flavus and the Rhizopus sp., but they were not observed in culture filtrates from intact (nondisrupted) mycelia of the same strains. The interconversion activities of AF-B1 and AFL were not observed when the cell-free systems were preheated at 100 degrees C. These findings strongly suggest that the interconversion of AF-B1 and AFL is mediated by intracellular enzymes of A. flavus and the Rhizopus sp. In addition, the isomerization of AFL-A to AFL-B observed in culture medium was also found to occur by the lowering of the culture pH.