The in vitro reconstitution of a functional rough membrane active in protein synthesis

Rough endoplasmic reticulum was reconstituted from free polyribosomes and rough membrane stripped from its ribosomes by KCl and puromycin. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient, amino acid incorporation capacity and sensitivity towards protein synthesis inhibitors. When the reconstitution was done with double labelled polyribosomes ([³²P] polyribosomes, [³H] leucine labelling of nascent peptide chain before or after the attachment of the polyribosomes to the membrane) both labels banded together with the reconstituted rough membrane band. Hybrid rough membrane could be formed from rat liver stripped rough membrane and wheat germ ribosomes. This hybrid membrane could translate globin mRNA.     

GnRH systems of <Emphasis Type="Italic">Cichlasoma dimerus</Emphasis> (Perciformes,Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.This work was supported by grants UBACYT X-217, Conicet PIP 0539/R188; NIH-HD-29186 and Fogarty International Fellowship TW00086.     

Structure and stability of the CuII complexes with tandem repeats of the chicken prion

Prion protein (PrP) misfolding is one of the pivotal issues in understanding the rudiments of neurodegenerative disorders. The conformational change of mammalian cellular PrP to scrapie PrP is caused by an unknown agent, but there is reasonable evidence supporting the key role of copper ions in this process. The structure of the avian PrP was found to be very similar to the mammalian protein, although there is only 30% homology in the secondary structure. This work shows that copper ions are very effectively bound by hexarepeat fragments of chicken prion protein, although not as effectively as it was found in the case of mammalian protein. By means of potentiometric and spectroscopic techniques (nuclear magnetic resonance, circular dichroism, UV-vis, and electronic paramagnetic resonance), it was shown that Cu(II) ions coordinate to the chicken PrP hexapeptide domain in physiological pH via imidazole nitrogen donors of His residue(s). The binding pattern changes the structure of peptide involved, indicating a possible impact of Cu(II) ions in the biology and pathology of nonmammalian PrP, which could be similar to that found for mammalian PrP. The present study shows that, similar to the human prion octapeptide repeats, chicken prion hexapeptide repeats might bind copper ions in two different ways, depending on the number of repeats and metal/ligand molar ratio: (i) an intra-repeat coordination mode in which copper ion is chelated by His imidazole and deprotonated amide nitrogen in monomeric peptide and (ii) an inter-repeat coordination mode in which a polymeric peptide ligand (dimer and trimer) forms polyimidazole complexes that are very stable at physiological pH. Two proline residues inserted into the hexapeptide unit have a critical impact on the metal-binding ability.     

Histoenzymatische Untersuchungen über das Verhalten einiger Enzyme in Nebennieren nach lokaler und Ganzkörperbestrahlung

Zusammenfassung In dieser Arbeit beschÄftigen wir uns mit dem Verhalten der BernsteinsÄure-Dehydrogenase, der DPNH-Diaphorase, der Adenosintriphosphatase und der alkalischen Phosphatase in den Nebennieren gesunder Meerschweinchen sowie Versuchstieren, die einer Ganzkörper- und Lokalbestrahlung mit Dosen von je 400 r unterworfen wurden.Die Untersuchungen ergaben 24 Stunden nach Ganzkörperbestrahlung einen Anstieg, nach 4 Tagen ein Absinken der Atmungsfermente. Nach 11 Tagen waren die Reaktionen bei den Versuchstieren individuell verschieden.Die ATP-ase und die alkalische Phosphatase steigerte ihre AktivitÄt in den ersten Tagen nach der Bestrahlung, dann traf eine AktivitÄtssenkung ein.Bei lokaler Bestrahlung konnte in den Nebennieren ein AktivitÄtsanstieg der ATP-ase, alkalischen Phosphatase, DPNH-Diaphorase und eine AktivitÄtssenkung der BernsteinsÄure-Dehydrogenase festgestellt werden.Man nimmt an, da\ die VerÄnderungen die Folge einer Stress-Reaktion auf die Nebennieren sind, die noch durch extrasuprarenale Faktoren, wie Eiwei\spaltprodukte und elektrolytische Störungen dm Gesamtkörper beschleunigt werden.Herrn Professor Dr. A. Pischinger zum 65. Geburtstag gewidmet.     

Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

Conformation of dioxopiperazines,II. CNDO/2 quantum mechanical calculations on conformational preferences in cyclo (glycyl-L-alanyl), cyclo(glycyl-L-valyl), and both epimers of cyclo-di-(alanyl)

Conformational energy calculations, aimed at verification of the suitability of the semiempirical molecular orbital CNDO/2 method for conformational elucidations in cyclic dipeptides formed from amino acids with aliphatic side chains, have been carried out. The results obtained for four dioxopiperazines [DOP; cyclo(Glycyl-L-Alanyl), cyclo(Glycyl-L-Valyl), and both epimers of cyclo-di-(Alanyl)] point out very good agreement with experimental premises. The latter include (1) the preference of the cis-peptide bonds for being nonplanar, which results in twisted-boat conformations of the DOP ring; (2) greater stability of conformers with a side chain oriented axially over those with a side chain oriented equatorially; (3) the preference of cyclo(Gly-Val) for assuming a folded conformation with one of the side chain γ-methyl groups sticking over the DOP ring.     

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight

Planta - Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were...     

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid