Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-alpha A, B, C, D, F) and 2 molecular hybrids between them (IFN-alpha AD(Bg1II) and IFN-alpha DA(Bg1II)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-alpha B, C and F showed comparable antiproliferative activity which was greater than that of IFN-alpha A, D, AD(Bg1II) and DA(Bg1II). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-alpha A, D and AD(Bg1II) were observed to be more protective than IFN-alpha B, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.     

Identification of phospholipid platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human amniotic fluid and urine

Human amniotic fluid and fetal urine were examined for the presence of phospholipid platelet-activating factor (PAF). PAF was detected in lipid extracts of some samples of amniotic fluid obtained from women in labor but it was undetectable in samples of amniotic fluid obtained before the onset of labor. PAF was identified by chromatographic mobility, platelet aggregation and chemical modifications. LysoPAF was also present in amniotic fluid at higher concentrations than those of PAF. Both PAF and lysoPAF were identified also in newborn and adult urine.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

A system for coupled multiple-column separation of proteins

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Suppression of tumor promoter phorbolmyristate acetate-induced chromosome breakage by antioxidants and inhibitors of arachidonic acid metabolism

To gain insight into the mechanism of formation of chromosomal aberrations by the tumor promoter phorbolmyristate acetate (PMA) in human lymphocytes, we investigated the effect of antioxidants and inhibitors of arachidonic acid metabolism. Among the antioxidants bovine erythrocyte CuZn superoxide dismutase, glutathione peroxidase, mannitol (a scavenger of hydroxyl radicals), butylated hydroxytoluene and butylated hydroxyanisole were anticlastogenic while catalase and dimethylfuran (a scavenger of singlet oxygen) were inactive. These results show that the induction of aberrations by PMA occurs via indirect action, i.e. the intermediacy of superoxide and hydroxyl radicals. The following inhibitors of arachidonic acid metabolism were strongly anticlastogenic: the cyclo-oxygenase inhibitors indomethacin and flufenamic acid and the lipoxygenase inhibitor BN1015. Imidazole, nordihydroguaiaretic acid BN 1048 and 5,8,11,14-eicosatetraynoic acid were moderately active. The inhibitor of phospholipase A₂, fluocinolone acetonide, was also anticlastogenic.

We conclude that the oxidative metabolism of arachidonic acid is involved in the induction of chromosomal aberrations by PMA in human lymphocytes. However, because of the limited selectivity of these drugs, it is not yet possible to identify unambiguously the step(s) in the arachidonic acid cascade responsible for PMA clastogenicity.     

In vivo interactions of acrylonitrile with macromolecules in rats

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.     

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.