Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.     

Substance use disorders: a comprehensive update of classification,epidemiology, neurobiology,clinical aspects,treatment and prevention

Substance use disorders (SUDs) are highly prevalent and exact a large toll on individuals’ health, well-being, and social functioning. Long-lasting changes in brain networks involved in reward, executive function, stress reactivity, mood, and self-awareness underlie the intense drive to consume substances and the inability to control this urge in a person who suffers from addiction (moderate or severe SUD). Biological (including genetics and developmental life stages) and social (including adverse childhood experiences) determinants of health are recognized factors that contribute to vulnerability for or resilience against developing a SUD. Consequently, prevention strategies that target social risk factors can improve outcomes and, when deployed in childhood and adolescence, can decrease the risk for these disorders. SUDs are treatable, and evidence of clinically significant benefit exists for medications (in opioid, nicotine and alcohol use disorders), behavioral therapies (in all SUDs), and neuromodulation (in nicotine use disorder). Treatment of SUDs should be considered within the context of a Chronic Care Model, with the intensity of intervention adjusted to the severity of the disorder and with the concomitant treatment of comorbid psychiatric and physical conditions. Involvement of health care providers in detection and management of SUDs, including referral of severe cases to specialized care, offers sustainable models of care that can be further expanded with the use of telehealth. Despite advances in our understanding and management of SUDs, individuals with these conditions continue to be stigmatized and, in some countries, incarcerated, highlighting the need to dismantle policies that perpetuate their criminalization and instead develop policies to ensure support and access to prevention and treatment.     

Enhanced susceptibility to cetyl fluoride (CF) and cetyl fluoroacetate (CFA) in DDT-resistant insects

Two compounds showing negative correlation to DDT-resistance in larvae of Musca domestica were found, namely cetyl fluoride (CF) and cetyl fluoroacetate (CFA). Larvae of the DDT-resistant housefly strains K₁ and TP were affected more strongly by CFA incorporated in the breeding medium than larvae of the normal reference strains Sv and S-Rome. The same held true for CF, except that the effect on strain TP was doubtful. The chlordane-resistant, strain R-Sard. had a normal response to CF and CFA. In larvae of several DDT-resistant, laboratory-developed strains of Anopbeles atroparvus, negative correlation to resistance was noted for CF, but not for CFA and cetyl cyanide. The effect of CF applied again only to DDT-resistance, not to dieldrin-resistance.No negative correlation to resistance was found in adults of houseflies and anophelines with regard to CF, CFA, cetyl chloroacetate, cetyl cyanide, cetyl thiocyanate and cetyl bromide.

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Zusammenfassung Cetylfluorid (CF) war gegen Hausfliegenlarven des Schweizer DDT-resistenten Stammes K₁ etwas giftiger als gegen die normalsensiblen Vergleichsstämme Sv und S-Rome; für den DDT-resistenten italienischen Stamm TP war diese Erscheinung jedoch fraglich. Cetylfluorazetat (CFA) wirkte etwas besser auf Larven der beiden DDT-resistenten Fliegenstämme als auf die der Vergleichsstämme. Gegen Anopheleslarven verschiedener DDT-resistenter Stämme, die alle von dem gleichen normalen Ausgangsstamm selektioniert wurden, war CF etwas besser wirksam, als gegen den normalsensiblen Ausgangsstamm. Dagegen konnte weder für CFA noch für Cetylzyanid erhöhte Empfindlichkeit bei resistenten Anopbeleslarven nachgewiesen werden. Die oben geschilderten Preferentialtoxizitäten für resistente Stämme bezogen sich nur auf DDT-Resistenz und waren für Chlordan-oder Dieldrin-Resistenz nicht zutreffend.Ausser dem in früheren Arbeiten geschilderten Fall (Cetylbromazetat bei adulten Hausfliegen) konnten keine neuen Beispiele für durch Resistenz induzierte, erhöhte Empfindlichkeit (Ascher 1958b, 1960) bei Adulttieren der Hausfliege und der Anopbelesmücke gefunden werden; es wurden zu diesem Zweck CF, CFA, Cetylchlorazetat, Cetylzyanid, Cetylrhodanid und Cetylbromid getestet, jedoch waren die Etgebnisse negativ. Verschiedene Cetyl-phosphorsäureester, Cetyl-polyäthylenaether und Alkylbrommalonate waren entweder überhaupt unwirksam oder wiesen bei vorhandener schwacher Wirksamkeit weder gegen Larven noch gegen Adulttiere resistenter Stämme eine preferentielle Toxizität auf.

     

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation.

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

Mapping of transfer RNA genes on tobacco chloroplast DNA

Tobacco chloroplast tRNAs have been purified by two-dimensional polyacrylamide gel electrophoresis, identified by aminoacylation, labelled at their 3-end and hybridized to tobacco chloroplast DNA restriction fragments, in order to establish a tRNA gene map. These hybridization studies have revealed the localization of at least seven genes in each inverted repeat region, a minimum of 22 tRNA genes in the large single copy region and one tRNA gene in the small single copy region. Comparison of the tobacco chloroplast tRNA gene map to that of maize shows many similarities, but also some differences suggesting that DNA sequence rearrangements have occurred in the chloroplast genome during evolution.     

Oxidation of methyl derivatives of pteridin-4-one, lumazine and related pteridines by bovine milk xanthine oxidase.

1. Pteridin-4-ones, methylated at nitrogen or carbon, N-methylated lumazines and related oxopteridines were studied as substrates of a highly purified bovine milk xanthine oxidase (xanthine : oxygen oxidoreductase, EC 1.2.3.2). 2. The enzyme can oxidise at high rates both uncharged and anionic substrates. Variation of enzymic activity with pH is mainly due to pH-dependent changes in the active enzymic center. 3. Milk xanthine oxidases at different stages of purification convert pteridin-4-one into the 4,7-dione (compound 13 in this article). 4. Methylation at C-6 in the pyrazine moiety enhances enzymic attack at C-2 in the pyrimidine ring. N-Methylation may increase or reduce rates of oxidation. 5. For oxidation at C-2, the most favorable form of the substrate bears a double bond at C(2) = N(3). Attack at C-7 is enhanced strongly in structures bearing a double bond at C(6) = C(7). 6. In general, pteridines react with xanthine oxidase as non-hydrated molecules. However, oxidation of 8-methyllumazine at C-7 may take place by dehydrogenation of the 7-CHOH group of the covalently hydrated molecule.