A monoclonal antibody that inhibits opioid binding to rat brain membranes

To understand the structure-function relationship and to probe the molecular characteristics of the purified opioid receptor, monoclonal antibodies (mab) were raised against a purified opioid receptor protein. After intensive screening of almost 1500 hybridoma cell lines, only 7 clones were shown to have very high immunoreactivity against the purified receptor. Moreover, out of these 7 clones, only 2, 3B4F11 and 3A27G, were found to inhibit the ligand binding property of the mu-opioid receptor. The mab 3B4F11 was found to inhibit 3H-diprenorphine binding to the purified receptor in a dose dependent manner with a maximal inhibition of 100% achieved with 20 micrograms of the antibody. Likewise, Fab fragments prepared from the mabs 3B4F11 inhibited 3H-diprenorphine binding to P2 membranes in a dose-dependent manner. In addition, it was found that the binding of 3H-DAGO, 3H-DPDPE and 3H-EKC was inhibited with approximately equal potency, suggesting that the Fabs prepared from the mab 3B4F11 interact with all 3 receptor types.     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

Intermediary carbon metabolism of Azospirillum brasilense.

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.     

Carbohydrate binding activities ofBradyrhizobium japonicum: IV. Effect of lactose and flavones on the expression of the lectin,BJ38

BJ38 is a galactose/lactose-specific lectin (M _r 38000) found at one pole ofBradyrhizobium japonicum. It has been implicated in mediating the adhesion of the bacteria to soybean roots, leading to the establishment of a nitrogen-fixing symbiosis. When the ligand lactose is added to cultures of the bacteria for at least 1 h prior to harvesting the cells for BJ38 isolation, the yield of the protein was found to be elevated in a dose-dependent fashion. Half maximal stimulation was observed at 50 µm; the effect was saturated at 1mm, where a 10-fold higher yield of BJ38 was obtained. Saccharides with a lower affinity for BJ38 than lactose yielded a correspondingly smaller induction effect when compared at a concentration of 1mm. The higher level of BJ38 induced by lactose is also manifested by an elevated amount of BJ38 detectable at the cell surface and by a higher number ofB. japonicum cells adsorbed onto soybean cells. Surprisingly, the induction of BJ38 expression seen with lactose was also observed with certain, but not all, flavonoids that induce thenod genes of the bacteria; genistein mimicked the induction observed with lactose, whereas luteolin failed to stimulate BJ38 production.     

Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice.

Transgenic mice that express the hepatitis B virus core protein were used to examine factors that influence the intracellular localization of nucleocapsid particles in the primary hepatocyte in vivo. In this model, viral nucleocapsid particles are strictly localized to the nucleus of the hepatocyte except when the nuclear membrane dissolves during cell division, at which time they enter the cytoplasm. The cytoplasmic nucleocapsid particles do not reenter the nucleus, however, when the nuclear membrane re-forms after cell division. The data support the notion that nucleocapsid particles can form de novo within the nucleus, and they suggest that performed nucleocapsid particles cannot be transported across the intact nuclear membrane in either direction. The results imply that nucleocapsid disassembly is probably required for entry of the hepadnaviral genome into the nucleus, and they question the role of the intranuclear viral nucleocapsid particle during the viral life cycle.     

Alginate-chitosan coacervation in production of artificial seeds

Survival of secondary embryoids of winter oilseed rape (Brassica napus ssp. oleifera cv. Primor) has been used as an assay for the development of artificial seeds involving complex coacervation of alginate (polyanion) with chitosan (polycation). Germination frequency of 100% was achieved for encapsulated embryoids when alginate formed the inner matrix and chitosan the outer layer. When the matrix makeup was reversed, there was no germination of embryoids. The artificial seeds produced were hardened in dilute alkaline solutions of NaOH and Ca(OH)(2). An optimum setting time could be selected based on a quantitative measurement of resistance of hardened capsules to compression and the germination frequency of the encapsulated embryoids. (c) 1993 John Wiley & Sons, Inc.     

Comparison of the Molecular Forms of the Kex2/Subtilisin-Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl-Terminus Truncation and Membrane Association

Abstract: The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full-length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full-length 86-kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl-terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane-associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86-kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus-truncated 64-kDa form.     

A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library

Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.