Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation ofq84t74420m5m73/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding ofq84t74420m5m73/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation ofq84t74420m5m73/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield ofq84t74420m5m73/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers ofq84t74420m5m73/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecularq84t74420m5m73/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-sheets.     

Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

The action of thyroid hormones on the expression of the mitochondrial ATP synthase q5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit gene (ATPsynq5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsynq5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5q5/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> upstream region of ATPsynq5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> gene were unresponsive to T₃ when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T₃ in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsynq5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> expression occur through indirect mechanisms.     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁-adrenoceptors (qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁AR) and qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂-adrenoceptors (qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR) mediate positive inotropic effects but that only qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁ AR and qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-AR comprise around 2/3 of qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁AR and 1/3 of qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR, whether or not qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR, we used the qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 qj1k888q74127xm/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 qj1k888q74127xm/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR than for qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">- 1 AR; the binding to qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR but not to qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">- BAR was reduced by GTPyS (10 qj1k888q74127xm/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 qj1k888q74127xm/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M); (to activate both qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁AR and qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR maximally) and zinterol (10 qj1k888q74127xm/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₁AR and qj1k888q74127xm/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

α-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an q2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of q2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, q2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and q2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an q2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lipoic acid, coenzyme A, and pyruvate or q2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an q2/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-lipoic acid (K _0.5=1.4±0.8 mM) and lipoamide (K _0.5=0.9±0.3 mM) dependent manner.     

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of q688376v21033v5t/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)₇CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6-q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6-q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucans. Here we demonstrate that both the fungal and the bacterial q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucans are ligands of q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6-q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6-q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6-q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucans. Another type of cyclic q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucan, a 1,2-q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the q383l23jq264t052/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).     

Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely q3n8u56212v2623h/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by q3n8u56212v2623h/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid q3n8u56212v2623h/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid q3n8u56212v2623h/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 16q3n8u56212v2623h/xxlarge8758.gif" alt="ratio" align="BASELINE" BORDER="0">3 hexadecatrienoic acid - 18q3n8u56212v2623h/xxlarge8758.gif" alt="ratio" align="BASELINE" BORDER="0">3 q3n8u56212v2623h/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - q3n8u56212v2623h/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal q6224552337p328/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

Development ofgusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzymeq/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.