Solid-Supported Oligonucleotide Systems for Special Biomedical Applications

Abstract

The use of composite beads consisting of a 6 μm polystyrene core with 30 nm surface-bound silica particles to routine automatic oligodeoxynucleotide (ODN) synthesis is described.     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.     

Effect of acetaminophen on hepatic content and biliary efflux of glutathione disulfide in mice

The increased expiration of ethane and pentane by mice treated with hepatotoxic doses of acetaminophen suggests the possibility of oxidant mechanisms associated with the necrosis. However, studies in rats are not consistent with oxidant stress mechanisms causing the damage, because acetaminophen given to rats does not increase GSSG efflux, a sensitive index of intrahepatic oxidant stress. To compare the extent of oxidant stress generated by acetaminophen in mice versus rats, hepatic content and biliary efflux of GSSG and GSH in mice have been examined. Bile was collected from anesthetized male ICR mice before and after intraperitoneal administration of acetaminophen (325 mg/kg, 2.15 mmol/kg), t-butyl hydroperoxide (TBHP) (1.5 mmol/kg), diethyl maleate (400 mg/kg, 2.33 mmol/kg, in corn oil) or saline (control) and GSH and GSSG were measured by the enzymatic recycling method of Tietze. An increase in biliary GSSG efflux was produced by t-butyl hydroperoxide, but not by the other agents. Biliary GSH/GSSG ratios decreased in acetaminophen-treated animals, presumably reflecting the marked depletion of hepatic GSH, since a similar decrease was observed with non-hepatotoxic doses of diethyl maleate. The failure of acetaminophen to increase the hepatic content or biliary efflux of GSSG in ICR mice is not consistent with the view that oxidant stress mechanisms cause the damage, despite the increases in alkanes expired after acetaminophen administration in this specific animal model.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

Inhibition of early steps of de novo fatty-acid biosynthesis by different xenobiotica

The importance of the early steps of de novo fatty-acid biosynthesis is discussed in terms of rate-limiting enzymic reactions with respect to their inhibition by xenobiotics. The inhibitory spectra of allicin as an inhibitor of the acetyl-CoA-synthase, two classes of graminicides (cyclohexane-1,3-diones and aryloxyphenoxypropionic acids) as inhibitors of acetyl-CoA-carboxylase, and the two antibiotics cerulenin and thiolactomycin, which affect the condensing step in fatty-acid biosynthesis, are compared.     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

The course control system of beetles walking in an air-current field

The wind-orientated walk of carrion beetles Necrophorus humator F. was analysed under closed-loop conditions with a walking compensator and under openloop conditions with a paired tread wheel (Fig. 1).

The Photocycle of Bacteriorhodopsin in the Two-Dimensional Orthorombic Crystal Form of Purple Membrane

Laser flash photolysis and low-temperature absorption studies of the photocycle of orthorhombic purple membrane (o-PM) reveal the existence of the same K, L, and M intermediates as found in the native hexagonal purple membrane (h-PM). However, the 0 intermediate is missing in the o-PM. The absorption spectrum of the K intermediate of o-PM is blueshifted by ~15 nm relative to the K intermediate found in the hexagonal purple membrane. The decay relaxation time constants of M in the o-PM are higher by more than an order of magnitude than the corresponding relaxation time constants in the h-PM. Similarly to the h-PM, the decay of M depends on the pulse width of excitation. The time-independent anisotropy factor obtained in photoselection studies of the M intermediate demonstrates the complete immobility of bacteriorhodopsin (bR) within the o-PM matrix. The same anisotropy factor of 0.3 obtained for o-PM and for h-PM suggests that in both crystalline lattices the transition moment of the retinal chromophore has similar angles with the plane of the membrane. The dependence of the decay kinetics of M on its occupancy may suggest the existence of kinetic coupling between neighboring bR molecules.     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.     

Ergebnisse ornithologischer Beobachtungen auf Sardinien im Winter 1961/62

Zusammenfassung Das Vogelleben der Mittelmeerländer ist in der Zeit der Winterregen bisher kaum untersucht worden. Verf. teilt deshalb Beobachtungsergebnisse von der Insel Sardinien mit, die er vom 15. X. 1961–15. III. 1962 während eines Studienaufenthaltes machen konnte. Der genannte Zeitraum umfaßt Beginn und Ende der sardischen Regenzeit und wird als Winter bezeichnet.Insgesamt wurden 125 Vogelarten beobachtet, von denen 65 Jahresvögel, 56 Gastvögel und 4 Sommervögel Sardiniens sind.Bei den Jahresvögeln interessierte vor allem die Vertikalverbreitung. Im Vergleich mit der Vertikalverbreitung zur Brutzeit ergeben sich teilweise beträchtliche Verschiebungen der Höhengrenzen. Für 10 Jahresvögel wird auch die horizontale Verbreitung mit Punktkarten dargestellt. Weitere Kapitel behandeln Biotope, Schwarmbildung und Vergesellschaftung sowie den Gesang im Winter.Die Gastvögel werden nach ihrer Herkunft untersucht, wobei über 260 Ringfunde ausgewertet werden. Auch über Ankunft und Abzug, Massenschlafplätze und Wasservogelleben der Gastvögel wird berichtet.In einer tabellarischen Übersicht sind wichtige Daten aller beobachteten Jahres- und Gastvögel zusammengefaßt.Abschließend werden Aufgaben der weiteren ornithologischen Erforschung Sardiniens genannt, die vor allem in der Erkundung abgelegener Gebirgsteile und weiterer Beobachtertätigkeit in den Winter- und Sommermonaten bestehen.

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Summary The bird life of the Mediterranean countries has hardly been studied during the period of the winter rains. Therefore the author records of observations results made on Sardinia from the 15^th of October, 1961 to the 15^th of March, 1962 while studying. The period mentioned above comprises the beginning and end of the Sardinian rain period, which is termed winter.Alltogether 125 bird species have been observed, 65 of which were resident, 56 visitors and 4 Sardinian summer birds.Above all the elevation distribution of residents was of interest. Comparing the vertical distribution during the breeding period with that during the winter period considerable displacements in the elevation limits were shown. For 10 residents the horizontal distribution is also described by means of dotted maps. Further chapters deal with the environment, flock formation, socialization and the winter song.The breeding regions of the visitors has been studied from the records of more than 260 ringed birds. Reports of the visitors' arrival and departure, mass sleeping places and aquatic life was also given.In a tabulated survey important facts are summarized of all residents and visitors.Finally, problems of further ornithological studies on Sardinia are mentioned which are exploration of remote mountain parts and further observation during the winter and summer periods.

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Riassunto La vita degli uccelli del bacino mediterraneo è stata fino ad oggi poco esaminata durante la stagione delle piogge.Perciò l'autore comunica i risultati delle sue osservazioni, fatte nell'isola di Sardegna durante un soggiorno di studio dal 15. X. 1961 al 15. III. 1962.Questo periodo comprende l'inizio e la fine della stagione sarda delle piogge ed è chiamato inverno.In tutto furono osservate 125 specie, delle quali 65 sono stazionarie, 56 ospiti e 4 uccelli d'estate di Sardegna.La distribuzione verticale degli uccelli stazionari risulta di particolare interesse.Si riscontrano considerevoli spostamenti dei limiti d'altezza, confrontando la distribuzione verticale d'inverno e d'estate.Cartelli di punti mostrano la distribuzione orizzontale per 10 uccelli stazionari.Altri capitoli trattano di biotopi, formazioni di stormi, socializzazioni e canto d'inverno.La provenienza degli ospiti è studiata secondo la valorizzazione di più di 260 ritrovamenti di uccelli inanellati.Vengono trattati anche l'arrivo e la partenza degli ospiti, i posti per dormire in massa e la vita degli uccelli acquatici.Dati importanti su tutti gli uccelli, sia stazionari che ospiti, sono raccolti in un quadro sinottico.Infine si fa cenno ad altri problemi degli studi ornitologici in Sardegna, che consistono soprattutto nella esplorazione delle montagne remote ed in ulteriori osservazioni, sia nella stagione invernale che in quella estiva.

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Für seine ständige, wertvolle Hilfe und Beratung bei meiner ornithologischen Tätigkeit auf Sardinien danke ich Herrn Prof. Dr.G. Niethammer, für die Ermöglichung meines Studienaufenthalts Herrn Prof. Dr. Dr.R. Lehmensick sehr herzlich. Dank abstatten möchte ich auch den Universitäten Bonn und Cagliari sowie vielen sardischen Freunden, die mir auf vielfältige Weise behilflich waren. HerrnH. E. Wolters danke ich für die Durchsicht des Manuskripts.