Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken ß-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

Effect of phosphate residue of NADPH on the interaction between catalytic domains of a multifunctional polyketide synthetic enzyme 6-hydroxymellein synthase

Association of NADPH with the ketoreducing domain of 6-hydroxymellein synthase, a multifunctional polyketide synthetic enzyme of carrot, evoked the alternation of microstructure around the primary binding site of the co-substrates acetyl- and malonyl-CoAs, and this resulted in the marked decrease in Km value of the enzyme protein for acetyl-CoA. In contrast, the enzyme did not show the increase in the affinity to the substrate when NADPH was replaced by NADH. These results suggest that the phosphate residue attached to 2'-position of adenosyl moiety of NADPH molecule plays an important role in the co-operative interaction between these functional domains of the synthase. Copyright 1998 Elsevier Science B.V.

Regulation of catalytic activity of a multifunctional polyketide biosynthetic enzyme, 6-hydroxymellein synthase, by interaction between NADPH and phenylglyoxal-sensitive amino acid residue at the reaction center.

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification of the enzyme protein with phenylglyoxal. Appreciable differences in K(m) values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalytic activity of the NADPH-associated synthase was enhanced by the addition of free CoA, the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme. A marked deuterium isotope effect in the catalytic reaction of the native synthase-NADPH complex was appreciably decreased in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2'-position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.

Role of inward K+ channel located at carrot plasma membrane in signal cross-talking of cAMP with Ca2+ cascade.

Treatment of cultured carrot cells with dibutyryl cAMP or forskolin resulted in the appreciable decrease in extracellular K+ concentration. This decrease was found to be transient and the concentration of the ion in the culture medium restored to the original level within few minutes. The cAMP-induced decrease in K+ level in the medium was almost completely inhibited when carrot cells were incubated in the presence of K+ channel blockers, CsCl and tetraethylammonium chloride. Appreciable amounts of 45Ca2+ were discharged from 45Ca2+-loaded inside-out vesicles of carrot plasma membrane by the stimulation with cAMP, however, the release of the ion was significantly inhibited in the presence of the K+ channel blockers. The release of 45Ca2+ from the vesicles was also observed when K+ current was evoked with an ionophore, valinomycin, even in the absence of cAMP. These results suggest that the gating of some of the inward K+ channels located at plasma membrane of cultured carrot cells is controlled by cytoplasmic concentration of cAMP and the inward K+ current across the plasma membrane induced by the nucleotide elicits Ca2+ influx into the cells possibly by the activation of voltage-dependent Ca2+ channels.

Transacylase-like structure and its role in substrate channeling of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cell extracts.

6-Hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, lost the binding ability toward its co-substrates, acetyl- and malonyl-CoAs, by the treatment with the blocking reagents for serine-OH. In contrast, the enzyme retained the binding ability even when the two SH groups at the reaction center (cysteine-SH of the condensation enzyme and cysteamine-SH of acyl carrier protein) were blocked, and one substrate bound to the SH-blocked enzyme was readily replaced by the other. It appeared that the cysteine-SH accepted only acetyl moiety while cysteamine-SH was preferentially malonylated in the presence of both of the substrates. These results suggest that transacylase-like domain is involved in the structure of 6-hydroxymellein synthase as a common primary binding site of its co-substrates, and acetyl and malonyl moieties are properly channeled from their CoA esters to cysteine-SH and acyl carrier protein-SH via this domain, respectively.

Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells.

Increase in cytoplasmic cyclic AMP concentration stimulates Ca2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2+ influx. Cyclic AMP evoked discharge of Ca2+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 microM of free Ca2+, while Ca2+ lower than 0.1 microM did not affect the Ca(2+)-release. The Ca2+ flux across plasma membrane was restored from this Ca(2+)-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca2+ channel.

6-Hydroxymellein synthetase as a multifunctional enzyme complex in elicitor-treated carrot root extract.

Synthetic activity of a polyketide compound 6-hydroxymellein was induced in elicitor-treated carrot root tissues. The activity was significantly inhibited by an antiserum raised against the acyl carrier protein (ACP) of fatty acid synthetase, suggesting that the enzyme(s) for 6-hydroxymellein synthesis require(s) a functional unit similar to ACP. However, the synthetic activity was not stimulated by the addition of ACP purified from Escherichia coli and was not lost even after fractionation by gel-filtration chromatography. The active fraction obtained by gel-filtration (136 kDa) was subjected to immunoblot analysis, and a 128 kDa polypeptide in the fraction was found to cross-react with anti-ACP serum. These observations suggest that the biosynthesis of 6-hydroxymellein in carrot cells is catalyzed by an enzyme consisting of a single peptide chain.

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.

Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract.

A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells.

Dissociation of dimeric 6-hydroxymellein synthase, a polyketide biosynthetic enzyme in carrot cell extracts, with loss of keto-reducing activity.

6-Hydroxymellein synthase, an inducible polyketide biosynthetic enzyme in carrot cell extracts, is composed of two identical subunits, and the homodimer is dissociated to monomeric peptides under high-ionic-strength conditions with loss of the synthase activity. Appreciable radioactivities were associated with the synthase proteins when the monomer enzyme was incubated with the radiolabeled substrates, acetyl-coenzyme A (CoA) and malonyl-CoA. Therefore, it appeared that the synthase does not lose the ability of binding the substrate even after the dissociation to monomers. The monomeric synthase liberated triacetic acid lactone as the derailment product instead of 6-hydroxymellein from the enzyme-attached triketomethylene chain which is the immediate precursor of an NADPH-dependent keto-reducing reaction involved in 6-hydroxymellein biosynthesis. These observations strongly suggest that the monomeric synthase retains the ability of ketomethylene chain elongation by the condensation of acyl-CoAs, but is lacking in an NADPH-dependent keto-reducing activity toward the triketide intermediate. Results obtained in the present experiments imply that the catalytic domain of acyl-CoA condensation is able to associate with that of keto reduction, possibly belonging to another subunit, only in the homodimeric structure to organize the multicatalytic reaction center.

Effect of NADPH-associated keto-reducing domain on substrate entry into 6-hydroxymellein synthase, a multifunctional polyketide synthetic enzyme involved in phytoalexin biosynthesis in carrot.

6-Hydroxymellein synthase is a polyketide biosynthetic enzyme induced in carrot cells which is organized as a homodimer composed of multifunctional subunits. The synthase liberates triacetic acid lactone, instead of 6-hydroxymellein, as a derailment product when the keto-reducing reaction at the triketide intermediate stage is interrupted. However, the efficiency of the triacetic acid lactone-forming reactions is markedly lower than that of the normal reaction, and the kinetic analyses have revealed that the affinity of the enzyme protein for acetyl-CoA is appreciably reduced in the abnormal reactions. It is assumed that the interaction of the NADPH-associated keto-reducing domain with a putative primary binding site(s) of the acyl-CoA in the enzyme structure affects the entry of the starter unit into the protein. The present finding should provide an example of the novel class of "subunit communication" of multimer enzymes.

Stimulation of calcium influx and calcium cascade by cyclic AMP in cultured carrot cells.

Treatment of cultured carrot (Daucus carota L.) cells with activators of adenylate cyclase, forskolin, and cholera toxin induced the biosynthesis of an antifungal isocoumarin, 6-methoxymellein, in the cells. Addition of dibutyryl cyclic AMP to carrot cell culture also stimulated the accumulation of the compound. The cyclic AMP-evoked 6-methoxymellein production was significantly depressed in the presence of certain inhibitors of calcium cascade such as Ca2+ channel blockers and inhibitors of calmodulin-dependent processes. In dibutyryl cyclic AMP- and forskolin-treated carrot cells, increase in cytosolic Ca2+ concentration was observed as monitored by the fluorescent calcium indicator fluo-3. Cyclic AMP-dependent Ca2+ influx into carrot cells was also confirmed with Ca(2+)-loaded vesicles prepared from the plasma membrane-rich fraction of the cells. Transient increase in Ca(2+)- and Ca2+/calmodulin-dependent protein kinase activity but not cyclic AMP-dependent protein phosphorylation was detected in the cells of high cyclic AMP concentration. Results obtained in the present work suggest that the increase in cyclic AMP content in carrot cells induces Ca2+ influx across plasma membrane without activating cyclic AMP-dependent protein kinase which, then, stimulates calcium cascade in the cells.

Role of reducing co-factors in catalytic reactions of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells.

6-Hydroxymellein (6HM) synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, is capable of catalyzing the acyl-CoA condensation and the ketoreduction in the presence of the nucleotide reducing co-factors. Although free CoA at high concentrations functioned as the activator of the NADPH-dependent 6HM formation, the compound exhibited an appreciable inhibitory activity toward the reaction mediated by NADH. CoA showed a potent inhibitory activity against substrate entry into the reaction center of the NADH-associated enzyme while, in the presence of NADPH, the compound slightly inhibited the formation of the acylated enzyme. The catalytic rate of the synthase was appreciably decreased when NADPH was replaced by the deuterium-labeled compound, however, the kH/kD value was markedly reduced if NADH and [D]NADH were employed as the reducing co-factors. These results suggest that the phosphate group attached to 2'-position of the adenosyl moiety of NADPH associated with the ketoreducing domain of 6HM synthase plays an important role in the regulation of the enzyme activity.

Breakdown of Phosphatidylinositol during the Elicitation of Phytoalexin Production in Cultured Carrot Cells.

Elicitor-induced production of the phytoalexin, 6-methoxymellein, in cultured carrot cells was appreciably depressed by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. An inhibitor of Ca(2+)-phospholipid dependent protein kinase (protein kinase C), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, also inhibited the phytoalexin production in carrot. Both phorbol ester and synthetic diacylglycerol, activators of protein kinase C, showed an ability to induce 6-methoxymellein production even in the absence of elicitor. Phosphatidylinositol-degrading phospholipase activity increased in elicitor-treated carrot cells without a notable lag, and a product of this reaction, inositol trisphosphate, appeared to increase in parallel with the phospholipase activity. These results suggest that breakdown of phosphatidylinositol takes place in the elicitor-treated carrot cells. The messengers liberated from the phospholipid in the plasma membrane may participate in the elicitation process by controlling the activity of protein kinase C-like enzyme(s) and Ca(2+)-mediated processes including calmodulin.

Carrot phytoalexin alters the membrane permeability of Candida albicans and multilamellar liposomes.

The biochemical basis for the antimicrobial effect of the carrot phytoalexin 6-methoxymellein (6-MM) was examined. At fungistatic concentrations 6-MM retarded the ability of Candida albicans to incorporate radioactive thymidine, uridine and leucine into biopolymers. When C. albicans was incubated with 6-MM, 260-nm-absorbing materials and 3H-labelled compounds leaked from the cells. The inhibitory effects of 6-MM on cell growth and membrane functions were, however, reduced as the concentration of divalent metal cations added to the medium was increased. 6-MM interacted with multilamellar liposomes constituted from phosphatidylcholine, cholesterol and dicetyl phosphate, or from phosphatidylcholine only, resulting in the release of glucose trapped in these liposomes. These results suggest that 6-MM exerts its toxic effects on susceptible cells as a result of its interaction with their membranes and disturbance of membrane-associated functions.

Role of reducing co-factor in cerulenin-insensitivity of 6-hydroxymellein synthase in carrot cell extract.

The activity of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, was not inhibited by cerulenin in the presence of NADPH. However, cerulenin showed a marked inhibitory activity to the synthase if the reducing co-factor was omitted from the assay mixture. The synthase was also sensitive to the antibiotic even in the presence of NADPH when the acyl condensation site and the reducing domain at the reaction center of the enzyme were dissociated under the high ionic strength condition. In addition, the synthase activity was appreciably inhibited when NADH was employed instead of NADPH. These observations strongly suggest that a phosphate group attached to 2'-position of adenosyl moiety of NADPH molecule plays an important role in the apparent insensitivity of 6-hydroxymellein synthase toward cerulenin.

Derailment product in NADPH-dependent synthesis of a dihydroisocoumarin 6-hydroxymellein by elicitor-treated carrot cell extracts.

Synthetic activity of 6-hydroxymellein, the immediate precursor of carrot phytoalexin 6-methoxymellein, from acetyl-CoA and malonyl-CoA was induced in carrot cell extracts when the root disks were treated with CuCl2 or oligogalacturonide elicitor. These elicitors showed specific inducing activity of phytoalexin production and did not affect fatty acid synthesis in carrot tissues which may share some common properties with 6-hydroxymellein biosynthesis. 6-Hydroxymellein production was an NADPH-dependent process and, in the absence of the reagent, triacetic acid lactone was produced as a derailment product of the reaction process. This finding suggested that the reduction of the double bond at the 3,4-position of the phytoalexin takes place during the elongation of the poly(oxomethylene) chain. This NADPH-dependent reduction seems to occur at the triacetate stage before the condensation of the third malonyl-CoA as the conversion of carbonyl to hydroxyl group.

Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells.

Biosynthetic activity of carrot phytoalexin 6-methoxymellen was induced in cell suspension culture by the treatment with oligogalacturonide elicitor; however, the elicitor-induced activity appreciably reduced in the presence of suramin, a potent inhibitor of GTP-binding proteins. In contrast, addition of G-protein activators, such as mastoparan or GTP-gamma-S, to carrot cell culture triggered 6-methoxymellein production even in the absence of uronide elicitor. An appreciable GTPase activity was found in purified plasma membrane of cultured carrot cells, and the hydrolytic activity was significantly increased by the addition of elicitor. Carrot plasma membrane was capable of associating with GTP-gamma-S, and the binding ability was markedly increased in the presence of elicitor. However, the binding activity markedly decreased when the membrane preparation was pre-incubated with GTP but not with ATP. These observations strongly suggest that a certain GTP-binding protein located at plasma membrane of cultured carrot cells plays an important role in the oligogalacturonide elicitor-induced 6-methoxymellein production.