Cardiotoxicity of Jamesoni's mamba (Dendroaspis jamesoni) venom and its fractionated components in primary cultures of rat myocardial cells

Summary Primary cultures of spontaneously beating myocardial cells isolated from neonatal rat hearts were used to screen the cardiotoxic effects of Jamesoni's mamba (Dendroaspis jamesoni) venom and components isolated from the venom by gel filtration and ion exchange chromatography. Cardiotoxicity was evaluated on the basis of leakage of lactate dehydrogenase (LDH), changes in morphology, cell membrane lysis, cellular viability, and alterations in spontaneous beating activity. The whole venom caused dose- and time-dependent leakage of LDH, disruption of the cell monolayer, decreases in viability, and inhibition of beating activity. Gel filtration of the venom yielded eight fractions (DjI to DjVIII). DjI (30 μg/ml), DjII (20 μg/ml), and DjV (20 μg/ml) caused significant (P<0.001) leakage of LDH, extensive morphologic damage, and decreases in viability. At lower concentrations DjI to DjVIII caused progressive inhibition of spontaneous beating activity. The main fraction (DjV), which was the most toxic, was further separated into 14 polypeptides (Dj1 to Dj14) by ion-exchange chromatography using Bio-Rex 70. Based on the ability to induce LDH leakage, produce morphologic damage, lyse cell membranes, and arrest beating activity, four categories of polypeptides were identified: cardiotoxins, Dj1 and Dj2; cardiotoxinlike polypeptides, Dj3 to Dj8; less active membrane lytic polypeptides, Dj9 to Dj13; and membrane lytic polypeptide, Dj14. This study was supported in part by the Fulbright Scholar Program (1986–1987) and the Burroughs Wellcome Fund. D. A. is a Burroughs Wellcome Scholar in Toxicology.     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

Cadmium and mercury cause an oxidative stress-induced endothelial dysfunction

We investigated the ability of cadmium and mercury ions to cause endothelial dysfunction in bovine pulmonary artery endothelial cell monolayers. Exposure of monolayers for 48 h to metal concentrations greater than 3–5 μM produced profound cytotoxicity (increased lactate dehydrogenase leakage), a permeability barrier failure, depletion of glutathione and ATP and almost complete inhibition of the activity of key thiol enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In contrast, metal concentrations less than 1–2 μM induced increases in glutathione and thiol-enzyme activities with minimal changes in LDH leakage, barrier function and ATP content. At shorter incubation times (24 h or less), high concentrations of cadmium caused glutathione induction rather than depletion. Thus, oxidative stress and cytotoxicity induced by lower concentrations of the metal ions stimulate compensatory responses, including increased synthesis of glutathione, which presumably preserved the activity of key thiol enzymes, however these responses were not sustainable at higher metal ion concentrations. We conclude, while high concentrations of heavy metals are cytotoxic, lower concentration induce a compensatory protective response, which may explain threshold effects in metal-ion toxicity.     

Antioxidant activities of cultured <Emphasis Type="Italic">Armillariella mellea</Emphasis>

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus—Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition, and FeCl₂-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H₂O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1–100 μg/ml, the free radical scavenging activity was 73.7–92.1% for AM-H₂O, and 60.0–90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC₅₀ is 9.17 μg/ml for AM-H₂O and 7.48 μg/ml for AM-EtOH). In general, AM-H₂O showed a stronger antilipid peroxidation activity on different rat’s tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the antilipid peroxidation activity of AM-H₂O (IC₅₀–6.66 μg/ml) in brain homogenate was as good as IC₅₀–5.42 μg/ml. AM-H₂O (80.0 mg/g) possessed a significantly higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and antilipid peroxidation activities, especially the AM-H₂O in the brain homogenate. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 495–500. The text was submitted by the authors in English.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Some further observations on synergism between chloramphenicol and polymyxin B in relation toSalmonella bacteria

Summary The synergism between chloramphenicol and polymyxin B sulphate, earlier noticed using a replica technique, has been reexamined applying quantitative methods. Vital count experiments in suspensions ofSalmonella typhimurium in broth have revealed that addition of bacteriostatic concentrations of chloramphenicol in a range of 5–100 μg/ml to bacteriostatic concentrations of polymyxin (0.1–0.9 μg/ml) produce a strong bactericidal effect (synergism). Combinations of bactericidal concentrations of polymyxin (1 μg/ml and higher) with chloramphenicol also exert a very high degree of bactericidal activity, though no more than with polymyxin alone (no synergism). Chloramphenicol therapy sustained with polymyxin, may be of therapeutic value in the eradication of stubborn enteralSalmonella infections. With the technical assistance of MissM. J. Wisse.     

Growth and fruitbody formation ofGanoderma lucidum on media supplemented with vanadium, selenium and germanium

Responses of mycelia ofGanoderma lucidum to vanadium, selenium and germanium were examined over a wide range of concentrations (10–1, 120 μg/ml) in pure culture. Se and V were found to be highly toxic, but Ge was not toxic at the levels tested.Ganododerma lucidum cultivated on substrates of sawdust with V (30–80 μg/g) developed mature fruitbodies, but the bioaccumulation of V was quite low (2.5–7 μg/g in pileus, 12.5–21.5 μg/g in stipe and <1 μg/g in basidiospores). Se as Na₂SeO₄ labeled with⁷⁵Se was effectively taken up from substrates and accumulated in fruitbodies (mainly in pileus), then depleted by discharge of basidiospores. Ge as GeCl₄ labeled with⁷⁷Ge was easily uptaken and translocated into fruitbodies.     

Killing rate curve and combination effects of surfactin C produced from Bacillus subtilis complex BC1212 against pathogenic Mycoplasma hyopneumoniae

This study evaluated the killing rate as well as the antimycoplasmal effect of surfactins isolated from Bacillus subtilis complex BC1212, either alone or in combination with various antibacterials against Mycoplasma hyopneumoniae. Prior to the killing rate and the combination effect studies of surfactins, the minimal inhibitory concentrations (MICs) of various antibacterials and surfactins (consisting of surfactins A, B, C, and D) against M. hyopneumoniae were investigated. The MIC of all surfactins was found to be 64 μg/ml. The MICs of colistin (COL), norfloxacin (NFX), oxytetracycline (OTC), streptomycin (SM) and tiamulin (TIA) were >256, 0.063, 0.025, 4, and 0.015 μg/ml, respectively. In the killing rate curve studies, surfactin C at 2× MIC and 4× MIC concentrations was found to reduce viability by >3 log₁₀ c.c.u./ml within 2–4 h of incubation. Combination of surfactin C with other antibacterials showed additive interaction from the viewpoint of fractional inhibitory concentration (FIC) index of >0.5 but ≤2 as a borderline. Taking all results into consideration, surfactin C may be useful as a preventive or therapeutic adjuvant in the pathogenesis of mycoplasmal infection.     

Responses ofGanoderma lucidum to heavy metals

The lelvels of seven heavy metals and their toxicity towardGanoderma lucidum under various cultivation conditions were assessed. The contents of Mn, Cu, Zn, Cd, Hg, Pb and U in the fruitbodies of cultivatedG. lucidum, and sawdust substrates were determined to be at trace levels for U, 0.01–0.1 μg/g for Cd and Hg, and 1–5 μg/g for Pb, 10–120 μg/g for Mn, Cu and Zn. The effects of heavy metals, on the growth of mycelia ofG. lucidium in pure cultures were examined over a wide range of concentrations (10–3,000 μg/ml), and their toxicities were found to decrease in the order: Hg>Cd>Cu>U>Pb>Mn=Zn. The translocation and accumulation of Zn from contaminated substrates (at 10 μg/g) in fruitbodies were investigated by using⁶⁵Zn tracer, andG. lucidum was found to take up Zn with an efficiency of >60%, leading to accumulation of >100 μ/g, in fruitbodies and >80 μ/g Zn in basidiospores.     

Intensification of surfactant synthesis in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP⁺-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: Review of the literature

Voriconazole (Vfend™) is a new triazole that currently is undergoing phase III clinical trials. This review summarizes the published data obtained by NCCLS methods on the in vitro antifungal activity of voriconazole in comparison to itraconazole, amphotericin B, fluconazole, ketoconazole and flucytosine. Voriconazole had fungistatic activity against most yeasts and yeastlike species (minimum inhibitory concentrations [MICs] <2 μg/ml) that was similar or superior to those of fluconazole, amphotericin B, and itraconazole. Against Candida glabrata and C. krusei, voriconazole MIC ranges were 0.03 to 8 and 0.01 to >4 μg/ml, respectively. For four of the six Aspergillus spp. evaluated, voriconazole MICs (< 0.03 to 2 μg/ml) were lower than amphotericin B (0.25 to 4 μg/ml) and similar to itraconazole MICs. Voriconazole fungistatic activity against Fusarium spp. has been variable. Against F. oxysporum and solani, most studies showed MICs ranging from 0.25 to 8 μg/ml. Voriconazole had excellent fungistatic activity against five of the six species of dimorphic fungi evaluated (MIC₉₀s < 1.0 μg/ml). The exception was Sporothrix schenckii (MIC₉₀s and geometric mean MICs ≥ 8 μg/ml). Only amphotericin B had good fungistatic activity against the Zygomycetes species (voriconazole MICs ranged from 2 to >32 μg/ml). Voriconazole showed excellent in vitro activity (MICs < 0.03 to 1.0 μg/ml) against most of the 50 species of dematiaceous fungi tested, but the activity of all the agents was poor against most isolates of Scedosporium prolificans and Phaeoacremonium parasiticum (Phialophora parasitica). Voriconazole had fungicidal activity against most Aspergillus spp., B. dermatitidis, and some dematiaceous fungi. In vitro/in vivo correlations should aid in the interpretation of these results. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.     

Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives

A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 μg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 μg/ml; for another, 2.9 μg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 μg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8–21 μg/ml. Received May 1, 1998; accepted July 24, 1998.     

Flavodoxin from Wolinella succinogenes

A monomeric flavoprotein (18.8 kDa) was isolated from the soluble cell fraction of Wolinella succinogenes and was identified as a flavodoxin based on its N-terminal sequence, FMN content, and redox properties. The midpoint potentials of the flavodoxin (Fld) at pH 7.5 were measured as –95 mV (Fld_ox/Fld_s) and –450 mV (Fld_s/Fld_red) relative to the standard hydrogen electrode. The cellular flavodoxin content [0.3 μmol (g protein)^–1] was the same in bacteria grown with fumarate or with polysulfide as the terminal acceptor of electron transport. The flavodoxin did not accept electrons from hydrogenase or formate dehydrogenase, the donor enzymes of electron transport to fumarate or polysulfide. Pyruvate:flavodoxin oxidoreductase activity [180 U (g cellular protein)^–1] was detected in the soluble cell fraction of W. succinogenes grown with fumarate or polysulfide. The enzyme was equally active with Fld_ox or Fld_s at high concentrations. The K _m for Fld_s (80 μM) was larger than that for Fld_ox and for the ferredoxin isolated from W. succinogenes (15 μM). We conclude that flavodoxin serves anabolic rather than catabolic functions in W. succinogenes. Received: 15 May 1996 / Accepted: 21 June 1996     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Ectoenzymatic Activity and Uptake of Monomers in Marine Bacterioplankton Described by a Biphasic Kinetic Model

Abstract The kinetics of bacterial hydrolytic ectoenzymatic activity and the uptake of monomeric compounds were investigated in the Northwestern Mediterranean Sea. Aminopeptidase and α- and β-glucosidase activities were analyzed by using fluorogenic substrates at 15–22 concentrations ranging from 1 nM to 500 μM. Radiolabeled glucose and a mixture of amino acids were chosen as representatives of monomeric compounds, and the bacterial uptake rates (assimilation plus respiration) were determined over a wide range of substrate concentrations (from 0.2 nM to 3 μM). We found biphasic kinetics both for hydrolytic enzymes and uptake systems: high affinity enzymes at low concentrations of substrates (K _m values ranged from 48 nM to 2.7 μM for ectoenzymes and from 1.4 nM to 42 nM for uptake systems), and low affinity enzymes at high concentrations of substrates (K _m values ranged from 18 μM to 142 μM for ectoenzymes and from 0.1 μM to 1.3 μM for uptake systems). Transition between high and low affinity enzymes was observed at 10 μM for aminopeptidase and from 1 μM to 25 μM for glucosidases, and it was more variable and less pronounced for the uptake of glucose (40 nM–0.28 μM) and amino acids (10 nM–0.16 μM). Results showed that the potential rates of hydrolysis and uptake are tightly coupled only if the high affinity hydrolytic ectoenzymes and the low affinity uptake systems are operating simultaneously. Received: 5 March 1998; Accepted: 31 July 1998     

Enhancing effect of proteins derived from Xestia c-nigrum granulovirus on Mamestra brassicae nucleopolyhedrovirus infection in larvae of Autographa nigrisigna (Lepidoptera: Noctuidae) on cabbage

The insecticidal effect of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) and the enhancing activity of proteins derived from occlusion bodies (OBs) of Xestia c-nigrum granulovirus (GVPs) on the infectivity of MabrNPV were evaluated in a bioassay with second-instar larvae of Autographa nigrisigna (Walker) fed virus-applied cabbage plants. The lethal concentrations of MabrNPV achieving 50 and 95% mortality for A. nigrisigna were estimated to be 1.4 × 10⁵ and 3.1 × 10⁶ OBs/ml, respectively. When larvae were fed cabbage plants treated with MabrNPV and various concentrations of GVPs, the requisite concentration of GVPs achieving 95% mortality of A. nigrisigna was estimated to be 26.2–138.8 μg/ml in combination with 10⁴ OBs/ml MabrNPV and 8.46–24.09 μg/ml with 10⁵ OBs/ml MabrNPV. Increases in the concentration of MabrNPV or GVPs caused larval death at younger instars. A. nigrisigna has lower susceptibility to MabrNPV than M. brassicae and Helicoverpa armigera reported in Mukawa and Goto (J Econ Entomol 103:257–264, 2010). We estimated that the requisite concentration of MabrNPV for the control of A. nigrisigna was 10⁵ OBs/ml, which is a tenfold higher concentration than that for M. brassicae and H. armigera, with the increase achieved by adding 10 μg/ml GVPs.     

Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P glucose-6-phosphate - LDH lactate dehydrogenase - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone - PPDK pyruvate, orthophosphate dikinase - U unit of enzyme activity (mol/min)     

Electroporation-mediated transient gene expression in intact cells of sweetpotato

Summary Transient expression of the β-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotatoIpomoea batatas L. (Lam.) by electroporation. The influence of several factors including electric field strength, buffer composition, time course of transientGUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined onGUS gene expression (number of blue spots). MaximumGUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-μF capacitor in the presence of 20 μg DNA/ml and 8.3 μl NaCl (3M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study ofGUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 μg/ml in the EPR buffer had a slight effect on the expression frequency (from 20–110 blue spots, and 112 blue spots with 20 μg/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 μl PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue.     

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K _m (for UDP-glucose) 0.5–1.0 mM; there was also a minor activity with an unusually high K _m of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K _m values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E_L, E_M and E_H respectively). E_M was the single major contributor to extractable UDPGDH activity when assayed at 0.6–9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E_L-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E_H activity is not due to ADH. At 30 μM UDP-glucose, 20–150 μM UDP-xylose inhibited UDPGDH activity, whereas 5–15 μM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.