Biochemical and immunocytochemical characterization of monoclonal antibody TOP 35 raised against purified tonoplast from cress root

The vacuolar membrane, the tonoplast, is a proteinrich membranehitherto only few proteins in it have been identified. As anapproach for the identification of tonoplast proteins by monoclonalantibodies (MABs), purified tonoplast from cress roots (Lepidiumsativum L.) were used for immunization and plasma membranesas a control membrane to test the absence of antigen. The MABTOP 35 identified a glycoprotein of about 35 kDa in purifiedtonoplast of cress roots. Triton X-114 phase separation showedthat it was a hydrophobic integral membrane protein. In immunocytochemistrythe MAB TOP 35 strongly labelled the vacuolar membrane. Theabsence of cell wall or plasma membrane labelling by TOP 35indicates a distinct biosynthetic pathway of this protein tothe vacuolar membrane in plants. Key words: Immnocytochemistry, Lepidium sativum, monoclonal antibody, secretion, vacuole     

5-Hydroxy-3-Ethylamino-2-Oxindole Is Not Formed in Rat Brain Following a Neurotoxic Dose of Methamphetamine: Evidence that Methamphetamine Does Not Induce the Hydroxyl Radical-Mediated Oxidation of Serotonin

Abstract: Oxygen radicals have been implicated in the neurodegenerative and other neurobiological effects evoked by methamphetamine (MA) in the brain. It has been reported that shortly after a single large subcutaneous dose of MA to the rat, the serotonergic neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) is formed in the cortex and hippocampus. This somewhat controversial finding suggests that MA potentiates formation of the hydroxyl radical (HO^?) that oxidizes 5-hydroxytryptamine (5-HT) to 5,6-DHT, which, in turn, mediates the degeneration of serotonergic terminals. A major and more stable product of the in vitro HO^?-mediated oxidation of 5-HT is 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). In this investigation, a method based on HPLC with electrochemical detection (HPLC-EC) has been developed that permits measurement of very low levels of 5-HEO in rat brain tissue in the presence of biogenic amine neurotransmitters/metabolites. After intracerebroventricular administration into rat brain, 5-HEO is transformed into a single major, but unknown, metabolite that can be detected by HPLC-EC. One hour after administration of MA (100 mg/kg s.c.) to the rat, massive decrements of 5-HT were observed in all regions of the brain examined (cortex, hippocampus, medulla and pons, midbrain, and striatum). However, 5-HEO, its unidentified metabolite, or 5,6-DHT were not detected as in vivo metabolites of 5-HT. MA administration, in particular to rats pretreated with pargyline, resulted in the formation of low levels of N-acetyl-5-hydroxytryptamine (NAc-5-HT) in all brain regions examined. These results suggest that MA does not potentiate the HO^?-mediated oxidation of 5-HT. Furthermore, the rapid MA-induced decrease of 5-HT might not only be related to oxidative deactivation of tryptophan hydroxylase, as demonstrated by other investigators, but also to the inhibition of tetrahydrobiopterin biosynthesis by NAc-5-HT. The massive decrements of 5-HT evoked by MA are accompanied by small or no corresponding increases in 5-hydroxyindole-3-acetic acid (5-HIAA) levels. This is due, in part, to the relatively rapid clearance of 5-HIAA from the brain and monoamine oxidase (MAO) inhibition by MA. However, the loss of 5-HT without corresponding increases in its metabolites point to other mechanisms that might deplete the neurotransmitter, such as oxidation by superoxide radical anion (O₂^??), a reaction that in vitro does not generate 5-HEO or 5,6-DHT but rather another putative neurotoxin, tryptamine-4,5-dione. One hour after administration, MA evokes large depletions of norepinephrine (NE) throughout the brain but somewhat smaller decrements of dopamine (DA) that are restricted to the nigrostriatal pathway. Furthermore, MA evokes a major shift in the metabolism of both NE and DA from the pathway mediated by MAO to that mediated by catechol-O-methyltransferase. The profound and widespread effects of MA on the noradrenergic system, but more anatomically localized influence on the dopaminergic system, suggests that NE in addition to DA, or unusual metabolites of these neurotransmitters, might play roles in the neurodegenerative effects evoked by this drug.     

Herbicidal activity of phosphonic,phosphinic, and phosphonous acid analogues of phenylglycine and phenylalanine

A series of phosphonic, phosphinic, and phosphonous acid analogues of phenylglycine and phenylalanine was synthesized and tested as herbicides against Lepidium sativum and Cucumis sativus. Aminobenzylphosphonic acids exhibited notable herbicidal activity and thus represent a group of the most active herbicides found among aminophosphonic acids.     

Amino-modified silicate gels prepared by sol-gel processing as metal-ion sorbents

Two series of amino-modified silicate gels prepared by sol-gel processing were used to absorb Cu(II), Ni(II), Co(II), Mn(II) and Cr(III) from aqueous solutions. These easily prepared sorbents with various content of primary amino groups in series (A) or primary and secondary amino groups in series (AA) have reasonable stability. The gel composition, time and concentration dependence of the uptake of the metal ions by these materials were studied systematically. These materials would be further used as supports to disperse catalytically active phases by conventional wet chemical procedures. Apart from this they demonstrate potential for the preconcentration aid for transition metal analysis.     

MHC zygosity in the frog,Rana temporaria

The major histocompatibility complex (MHC) zygosity of the field-collected frogs, Rana temporaria, was detected by progeny testing. Groups of sibling tadpoles were grafted with intrafamilial tail-tip allografts and the ratio of rapidly rejected allografts to slowly rejected ones was estimated. Twenty-five percent of parental frogs appeared to be MHC homozygotes. Thus, MHC homozygosity in natural frog populations seems to be considerably higher than in wild mouse populations.     

Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver

Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained. The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared. Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed. Moreover, it was found that polypeptides with mol. wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000–33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively. A major difference in histone compostion of hamster hepatoma and liver concerns histones H2A and H1. Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed. Apparently chromatin-bound protease activity can be found in both fractions of chromatin.     

Interaction of metabolic inhibitors with actin fibrils

Summary The dependence of the arrangement of fibrillar actin in cultured endothelial cells on metabolic conditions was investigated with cellular elements derived from the heart of Xenopus laevis tadpoles. Either primary culture or an established cell line (XTH-2) were used in these studies The metabolic stage of the cells was influenced by inhibiting respiration and lactate production. The actin pattern was revealed either by indirect immunofluorescence or by tetramethylrhodaminyl (TRITC)-phalloidin fluorescence. Total block of energy supply causes in all cases a distinct loss of actin fibrils, while inhibition of respiration alone increases the variability of actin organization. In primary XTH cells but not in XTH-2 cells cyanide disintegrates most of the actin fibres during 3 h of treatment. This effect is independent of the inhibition of respiration, since actin gels prepared from skeletal muscle also undergo destruction in the presence of cyanide. It is concluded that the actin fibrils of the primary cells and the established line behave differently to changing metabolic conditions and to application of KCN.     

Polarity and competition between megaspores in the ovule ofOenothera hybrids

New data on the development of polarity in the ovules during megasporogenesis and early stages of embryo sac development inOenothera-hybrids are presented. It is confirmed that allOe. hookeri-hybrids show a strong tendency to form heteropolar tetrads, with the micropylar megaspore developing into an embryo sac. This preference is seen in the delay of the second meiotic division on the chalazal side, the absence of callose in the lateral wall of the micropylar megaspore, and the accumulation of starch in this megaspore. However, homopolar tetrads, chalazal preference, and ovules with two developing embryo sacs are also observed with considerable frequency. Quantitative data on the frequency of the different developmental types are compared with earlier genetic results about competition in the haplophase. There is sufficiently good agreement to support the hypothesis ofRenner that there is a correlation between the developmental processes in the megaspore tetrad and the genetic phenomena of competition in the haplophase.