Monoclonal antibodies with specific effects on partial activities of recA protein of Escherichia coli

recA protein of Escherichia coli promotes a wide variety of DNA reactions in vitro. Specific effectors of recA protein should be very useful in elucidating the mechanisms of these complex reactions. Six mouse hybridoma clones that secreted class G immunoglobulins specific to recA protein were obtained in three cell-fusion experiments. Five IgGs were purified by affinity chromatography. These monoclonal antibodies were characterized by examining their effects on the single-stranded DNA-dependent ATPase activity, negatively superhelical double-stranded DNA-dependent ATPase activity, and an activity in pairing negatively superhelical closed circular double-stranded DNA and homologous single-stranded DNA-fragments to form D-loops. These IgGs inhibited all, some, or one of these three activities, and from the spectra of their inhibitory effects they were classified into four groups. This classification suggests that each of the monoclonal antibodies binds to one of at least four antigenic determinants on recA protein and specifically inactivates one or more of the active centers on the protein. These monoclonal antibodies will be useful in analyzing the complex reactions promoted by recA protein.     

Insect lipid transfer particle catalyzes bidirectional vectorial transfer of diacylglycerol from lipophorin to human low density lipoprotein

Insect plasma lipid transfer particle (LTP) catalyzes vectorial net transfer of diacylglycerol (DAG) from Manduca sexta larval high density lipophorin (HDLp-L) to human low density lipoprotein (LDL) producing an LDL of lower density and lipophorin subspecies of higher density. At equilibrium, a stable DAG-depleted very high density lipophorin species (density = 1.25 g/ml) is formed. Electrophoretic analysis of the substrate and product lipoproteins showed that apoprotein exchange or transfer between human LDL and lipophorin did not occur during the lipid transfer reaction. Facilitated net transfer of cholesteryl ester, free cholesterol, and phospholipid occurred to a much lower extent than DAG net transfer, indicating that under these conditions, LDL serves as a sink for lipophorin-associated DAG. This reaction, therefore, provides a method whereby the mass of lipid associated with human LDL can be modified in vitro without alteration of its apoprotein component. The DAG content of LDL increased in a linear manner with respect to LTP concentration and time during the initial phase of the reaction, demonstrating the utility of this system as a quantitative assay method for LTP-mediated net DAG transfer. When [3H]DAG-labeled LDL was prepared and employed in transfer experiments with unlabeled lipophorin, labeled DAG was recovered in the HDLp-L fraction. The amount of labeled DAG recovered in the HDLp-L fraction was dependent on the ratio of LDL to HDLp-L in the reaction. Thus, in this system, LTP-mediated DAG redistribution is bidirectional, suggesting that the final equilibrium distribution of lipid may be dictated by the properties of potential donor/acceptor lipoproteins rather than by an inherent particle substrate specificity of LTP.     

Selective Increase in S-100β Protein by Aging in Rat Cerebral Cortex

Changes in the concentrations of nervous tissue-related proteins and their isoproteins, such as S-100 proteins (S-100 alpha and S-100 beta), enolase isozymes (alpha-enolase and gamma-enolase), and GTP-binding proteins (Go alpha, Gi2 alpha, and beta-subunits), were determined in the CNS of male rats of various ages (from 2 to 30 months old) by means of enzyme immunoassay. The weights of brains and the concentrations of soluble proteins in the cerebral cortex, cerebellum, and brainstem were constant during the observation period. The concentration of S-100 beta protein, which is predominantly localized in glial cells, increased gradually in the cerebral cortex with age; levels in the 25-month-old rats increased to approximately 150% of the levels in the young (2-month-old) rats. However, the S-100 beta concentrations in the cerebellum and brainstem were relatively constant, showing similar values in rats 2-30 months old. Levels of other proteins, including both neuronal (gamma-enolase and Go alpha) and glial (alpha-enolase and S-100 alpha) marker proteins, did not change significantly with age in the cerebral cortex, cerebellum, and brainstem. These results suggest that there is a close relation between the age-dependent changes of the CNS function and S-100 beta protein levels in the cerebral cortex.     

Detection of mRNAs correlated with proliferation of cells in suspension cultures of Catharanthus roseus

Gene expression correlated with proliferation was investigated in Catharanthus roseus (L.) G. Don. cells. Polyadenylated RNAs were isolated from cells in proliferative states or in nonproliferative states and the variations in the population and levels of mRNA were analyzed by in vitro translation followed by separation of the corresponding polypeptides by two-dimensional gel electrophoresis. Levels of two mRNAs increased in the actively cycling cells, while they were hardly detected in cells in stationary phase or in cells arrested by starvation of phosphate, sucrose or nitrogen. The molecular masses of the translated products of these two mRNAs were 53 and 60 kDa. No mRNAs were specifically synthesized in common among the various cultures of cells whose growth was arrested by nutrient starvation.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.     

Characterization of two novel pyruvylated glycosphingolipids containing 2'-aminoethylphosphoryl(-->6)-galactose from the nervous system of Aplysia kurodai.

Two novel acidic glycosphingolipids containing pyruvylated galactose were purified from the nervous tissue of Aplysia kurodai by successive Iatrobeads column chromatographies. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structures of these acidic glycosphingolipids, named F-9 and FGL-I, were determined to be: [3,4-O-(S-1-carboxyethylidene)]Gal beta 1-->3 GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2] (2-aminoethylphosphoryl 1-->6)Gal beta 1-->4Glc beta 1-->1ceramide and [3,4-O-(S-1-carboxyethylidene)] Gal beta 1-->3GalNAc alpha 1-->3(Fuc alpha 1-->2)(2-aminoethylphosphonyl-->6 Gal beta 1-->4Glc beta 1-->1ceramide, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, pyruvylated glycosphingolipids containing phosphoethanolamine in addition to or in place of 2-aminoethylphosphonate are present in the nervous system of Aplysia.     

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met¹), Ad(Met⁻¹), Ad(Met⁻¹²), and Ad(Met⁶). The shoulder numbers represent this site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met⁻¹), were purified from the cell lysate and the ratios of A₄₁₄-to-A₂₇₆ of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450_SCC reduction catalyzed by Ad(Met⁰) was about 60% or that by Ad(Met¹), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450_SCC would need to be considered.     

Effects of precocenes on vitellogenesis in the adult female tick,Ornithodoros moubata (Acari: Argasidae)

Vitellogenin (Vg) concentrations in the hemolymph and ovarian development were studied inOrnithodoros moubata after treatment with precocenes 1 (P1) and 2 (P2). Precocene was dissolved in acetone or DMSO and topically applied to the dorsal surface of ticks: (1) at adult ecdysis; (2) 24 h before engorgement; (3) immediately after engorgement; and (4) 24 h after engorgement. Subsequently, P1 and P2 were dissolved in olive oil and injected through the gonopore into the body cavity 24 h after engorgement. Vitellogenin concentration was measured on days 5 and 10 after engorgement and ovarian development was scored on day 10, 20 or 30. Oviposition was also recorded and the average weight of eggs laid by females was determined. No differences in concentration of Vg in the hemolymph occurred between the control ticks and ticks treated topically or by injection with P1 and P2. Precocene did not suppress the synthesis of Vg inO. moubata. However, oviposition was reduced in ticks that survived repeated treatment with high doses of P2 dissolved in acetone.     

Effects of eel atrial natriuretic peptide on NaCl and water transport across the intestine of the seawater eel

Summary Eel atrial natriuretic peptide inhibited the serosa-negative transepithelial potential difference and short-circuit current, accompanied by a decrease in NaCl and water absorption across the seawater eel intestine. Similar effects were obtained after treatment with N-terminally truncated eel atrial natriuretic peptide (5–27), indicating that N-terminal amino acids are not essential for the action of eel atrial natriuretic peptide. Although mammalian atrial natriuretic peptides also inhibited the short-circuit current, a 100-fold higher concentration was reuired to obtain the same effect as with eel atrial natriuretic peptide, indicating that eel atrial natriuretic peptide is 100 times as potent in eel intestine as the mammalian atrial natriuretic peptides. Similarly, in mammalian atrial natriuretic peptide, the four N-terminal amino acids had no significant effects. However, when the C-terminal tyrosine was removed, the potency of rat atrial natriuretic peptide was lowered. Compared with the effects of acetylcholine, serotonin and histamine, eel atrial natriuretic peptide was the most potent inhibitor, with 100% inhibition at 10^-7 M; 50% inhibition was obtained at 10^-2 M in acetylcholine, and 30% inhibition in serotonin (10^-5 M) and histamine (10^-3 M). These inhibitory effects of eel atrial natriuretic peptide were not diminished even in the presence of tetradoxin, and were mimicked by 8-bromoguanosine 3,5-cyclic monophosphate. Based on these results, structure-activity relationships of eel atrial natriuretic peptide and a possible mechanism of action of eel atrial natriuretic peptide are discussed.Abbreviations 8BrcGMP 8-bromoguanosine 3,5-cyclic monophosphate - eANP eel atrial natriuretic peptide - hANP human atrial natriuretic peptide - 5-HT 5-hydroxytryptamine creatine sulphate - I _sc short-circuit current - PD transepithelial potential difference - rANP rat atrial natriuretic peptide - R _t tissue resistance - TTX tetrodotoxin