Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Central alpha-activation by clonidine reduces plasma level of beta-endorphin in patients with essential hypertension

Whether peripheral beta-endorphin contributes to the antihypertensive action of clonidine was examined by measuring plasma levels of beta-endorphin-like immunoreactivity (beta EpLI) after acute administration of clonidine in patients with essential hypertension. Administration of clonidine (0.225 mg) in one dose significantly lowered blood pressure, decreased heart rate and reduced the plasma level of beta EpLI and ACTH, while the placebo had no effect on blood pressure, heart rate or plasma level of beta EpLI suggesting that peripheral beta-endorphin does not play a major role in the antihypertensive action of acute clonidine administration.     

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.     

Fine structural changes in the rat brain after local injections of gliotoxin, alpha-aminoadipic acid

Toxic effects of locally injected gliotoxin, L-alpha-aminoadipic acid, were examined in the adult rat brain at the fine structural level. Astrocytes were the only cell type in the corpus striatum that showed clear structural degeneration 1 to 3 days after the treatment. With a 7 day survival period, two major structural changes were detectable, including tremendous invasion of astrocytes and their processes, and an appearance of a large number of microglia around injection sites. Both the stereospecificity and cellular specificity were firmly established on the toxic effects of this chemical.     

Adenine-dependent growth of marine yeast,Rhodotorula glutinis var.salinaria under sodium chloride-hypertonic condition

Nitrate and ammonium were shown to alter the growth ofRhodotorula glutinis var.salinaria in saline and non-saline media. In saline medium in which ammonium was the sole nitrogen source, ammonium inhibited growth in the presence of molybdate ions. Detailed comparisons of the growth in saline and non-saline media when nitrate was supplemented in the presence of molybdate ions showed that differences in utilizability of purine bases of nucleic acid were responsible for the differences in growth,i.e. adenine increased the growth in such saline medium, but decreased it in non-saline medium. There was not such a specific requirement for adenine in saline medium in the presence of molybdate ions when nitrate was substituted for ammonium as the sole nitrogen source. It was suggested that adenine might provide the necessary skeleton of nucleic acid for serving nitrate reduction in saline medium.     

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.     

Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure

We have isolated cDNA clones encoding boar acrosin, a serine protease participating in the initial stage of fertilization, from boar testis lambda gt11 cDNA libraries. Nucleotide sequencing of the overlapping clones indicates that the composite cDNA inserts contain 1,391 base pairs coding for a 5'-untranslated region, an open reading frame, a stop codon, a 3'-untranslated region, and a poly(A)+ tail. A polyadenylation signal, AATAAA, is located 33 bases upstream from the start of the poly(A)+ tail. The amino acid sequence deduced from the cDNAs shows that boar acrosin is initially synthesized as a prepro-protein with a 16-residue signal peptide at the NH2 terminus. This signal sequence is followed by a 399-residue sequence corresponding to the acrosin zymogen. COOH-terminal sequence analysis of boar sperm 55-kDa proacrosin and its processed forms indicates that the mature acrosin molecule contains 322 amino acid residues in two polypeptide chains, a 23-residue light chain and a 299-residue heavy chain, with a combined molecular mass of 35,735 Da, and that the 55-kDa proacrosin molecule has 14-, 18-, and 43-residue segments as COOH-terminal extensions that are removed during proacrosin maturation. The COOH-terminal 43-residue segment is rich in proline residues, including an unusual repeat of 23 consecutive prolines. The deduced amino acid sequence of boar acrosin shows a high degree of identity with major portions of other serine proteases, including the active site region and the location of cysteine residues. We conclude that boar acrosin is synthesized as a single-chain polypeptide with the regions corresponding to the light and heavy chains covalently connected by two disulfide bonds, and that the single-chain molecule is autoactivated by cleavage of the Arg23-Val24 bond after removal of the COOH-terminal 14-residue segment, resulting in the formation of the light and heavy chains. This two-chain molecule is then converted to the mature enzyme by removal of the COOH-terminal 18- and 43-residue segments.     

Behavioral effects of benzodiazepine antagonists in chlordiazepoxide tolerant and non-tolerant rats

Rats were trained to respond under 3-min fixed-interval schedules of food presentation, and effects of the benzodiazepine-receptor ligands, flumazenil, 2-(4-methoxy-phenyl)-pyrazolo[4,3-c]quinolin-3(5H)-one (CGS 9895), 3-carbo-t-butoxy-beta-carboline (beta-CCtB), and beta-carboline-3-carboxylic acid ethyl ester (beta-CCE) were assessed before and after the induction of tolerance to chlordiazepoxide. Before daily administration of chlordiazepoxide, none of the antagonists produced appreciable effects on rates of responding up to doses of 32.0 mg/kg i.p. beta-CCE was the only antagonist studied at a higher dose (100.0 mg/kg i.p.), which decreased response rates. After 23 days of daily chlordiazepoxide administration (oral doses started at 10 and increased to 100 mg/kg/day by the 17th day), dose-effect curves for chlordiazepoxide were shifted to the right by about one-half log unit. Subjects were also more sensitive to the flumazenil, CGS 9895, and beta-CCtB, however, since these drugs produced only small effects in non-tolerant subjects, precise estimates of the degree of the shift in dose-effect curves could not be estimated. However, there were differences in the changes in the dose-effect curves induced by chlordiazepoxide tolerance. These results suggest differences in mechanism of action of antagonists in tolerant and non-tolerant subjects, and further that the sensitivity that is induced to antagonists in tolerant subjects is not conferred equally to all drugs having benzodiazepine antagonist activity.     

Application of a novel apparatus, the quartz chemical analyzer, to the determination of endotoxin in blood.

A novel apparatus called a quartz chemical analyzer (QCA) has been developed using a quartz crystal resonator. This apparatus measures sample viscosity changes based on resonant frequency changes of the quartz crystal. The apparatus was used to determine bacterial endotoxin concentrations by monitoring the gelation reaction of Limulus amebocyte lysate. The QCA determined endotoxin concentrations with good accuracy and reproducibility in the range of 0.001-3 EU/ml for endotoxin standard (JP XII). For endotoxin determination in human whole blood and plasma samples, the inhibitory reaction was eliminated by pretreatment of a fourfold dilution at 60 degrees C and incubation for 30 min. There are many advantages of the QCA method compared with the turbidimetric and chromogenic methods. For example, QCA can measure sample viscosity changes with high sensitivity and accuracy because QCA detects minor resonant frequency changes and the frequency data give a numerical value for easy quantitation. QCA can examine turbid samples, and the required quantities of samples and reagents are small, since the quartz crystal detects sample viscosity changes directly. The endotoxin determination time may be shortened by raising the reaction temperature, and QCA can detect other types of coagulation reactions.     

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.