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PubMed1991

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.).

The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall "loosening."

Journal Article↗
PubMed2000

Complex spatial responses to cucumber mosaic virus infection in susceptible Cucurbita pepo cotyledons.

Cucumber mosaic virus infection of its susceptible host Cucurbita pepo results in a program of biochemical changes after virus infection. Applying a spatial analysis to expanding infected lesions, we investigated the relationship between the changes in enzyme activity and gene expression. Patterns of altered expression were seen that could not be detected by RNA gel blot analysis. For all the host genes studied, there was a downregulation (shutoff) of expression within the lesion. In addition, two distinct types of upregulation were observed. The expression of heat shock protein 70 (HSP70) and NADP(+)-dependent malic enzyme (NADP-ME) showed induction in apparently uninfected cells ahead of the infection. This response was more localized than the upregulation exhibited by catalase expression, which occurred throughout the uninfected regions of the tissue. The experiments showed that virus infection induced immediate and subsequent changes in gene expression by the host and that the infection has the potential to give advance signaling of the imminent infection.

Catalase↗
PubMed2000

Crystallization and preliminary crystallographic study of cucurmosin, a ribosome-inactivating protein from the sarcocarp of Cucurbita moschata.

Cucurmosin, a ribosome-inactivating protein purified from pumpkin, the sarcocarp of Cucurbita moschata, has been crystallized using polyethylene glycol as a precipitant. The crystals belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 41.91, b = 59. 48, c = 98.78 A. There is one molecule in the asymmetric unit. The diffraction data to 3.0 A resolution were collected on a MAR Research image-plate detector.

Crystallization↗
PubMed2003

Crystallization and preliminary X-ray crystallographic studies of mavicyanin from Cucurbita pepo medullosa.

Mavicyanin isolated from Cucurbita pepo medullosa is a glycosylated protein containing a single polypeptide chain of 109 amino-acid residues and is a member of the phytocyanin subclass of cupredoxins. Non-glycosylated recombinant mavicyanin, which was expressed in Escherichia coli, was crystallized by the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant at pH 5.5. The crystals belonged to the hexagonal space group P6(1) (or P6(5)), with unit-cell parameters a = 64.0, c = 245.0 A, four molecules per asymmetric unit and a solvent content of 59%. X-ray diffraction data were collected to 1.6 A resolution. To solve the structure of mavicyanin, the MAD method as well as a Patterson search method using the structure of stellacyanin as a starting model are presently being utilized.

Ammonium Sulfate↗
PubMed2001

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.

Circular Dichroism↗
PubMed1979

Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties.

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.

Amino Acids↗
PubMed1979

Mavicyanin, a blue copper protein from Cucurbita pepo medullosa. Purification and characterization.

1. The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. 2. Mavicyanin contains one type-1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (epsilon = 5000 M-1 cm-1/Cu, A280/A600 = 8.0 +/- 0.5, A600/A403 = 7.0 +/- 0.25, maximum of fluorescence emission at 335 nM. 3. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz = 2.287, gy = 2.077, gx = 2.025, Az = 3.5 mT, Ay = 2.9 mT and Ax = 5.7 mT. 4. Like other simple type-1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicyanin is readily reduced by hydroquinone or L-ascorbic acid. Its midpoint potential E'm was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.

Amino Acids↗
PubMed1983

Subunit structure and interactions of the phloem proteins of Cucurbita maxima (pumpkin).

The two major proteins from the phloem exudate of Cucurbita maxima (pumpkin), PP1 and PP2, were stable in the absence of reducing agents after modification of their accessible cysteine residues with iodoacetamide. This permitted their purification without precautions to prevent oxidation. PP2, a lectin specific for oligomers of N-acetyl-D-glucosamine, was shown by sedimentation-equilibrium ultracentrifugation to be a dimer of Mr of 48000. Neither dithiothreitol nor tri-(N-acetyl-D-glucosamine) altered this value. The constituent polypeptides were linked by two buried disulphide bridges. PP2 behaved aberrantly on gel-filtration on both Sephadex and Bio-Gel unless tri-(N-acetyl-D-glucosamine) was added to the elution buffer; the Mr was then measured as 46000. Other proteins which bind oligomers of N-acetyl-D-glucosamine are also retarded on gel-filtration. Soluble phloem filaments were prepared by collection of exudate into deaerated buffer containing iodoacetamide but no reducing agent. Oxidative gellation of the filaments was prevented by rapid modification of their many accessible cysteine residues, and is assumed to have maintained the degree of polymerisation found in vivo. Those disulphide bridges which were present allowed the incorporation of approximately 60% of the PP1 and 80% of the PP2 into polymeric material. It is concluded that PP1 and PP2 are both structural proteins present in the filaments observable in vivo. PP2 had an elongated binding-site for oligomers of N-acetyl-D-glucosamine. It is suggested that this lectin immobilises bacteria and fungi to the cross-linked filaments which seal wounded phloem sieve-tubes, and thus maintains sterility.

Chemical Phenomena↗
PubMed1996

Purification, characterization and subcellular localization of a type-1 ribosome-inactivating protein from the sarcocarp of Cucurbita pepo.

The flesh of the fruit of Cucurbita pepo contains a type-1 ribosome-inactivating protein (RIP), which we named pepocin. Pepocin was purified to apparent homogeneity by acid fractionation, ion-exchange chromatography and adsorption chromatography. The protein was found to have a molecular mass of 26 kDa and a pI of about 9.9. It does not contain glycosidic linkages. The protein inhibits protein synthesis in a rabbit-reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 15.4 pM, and depurinates 28S rRNA in the ribosomes of the lysate in a manner identical to that of ricin A-chain and other RIP. The enzyme is also active on wheat-germ ribosomes and on Escherichia coli ribosomes. The sequence of the N-terminal 20 amino acids of the protein reveals a close relationship to other RIP. Immunoelectron-microscopic localization of pepocin in the sarcocarp shows that the protein is predominantly localized in intercellular spaces. In addition, the immunolocalized signals are observed in leaf intercellular spaces.

Amino Acid Sequence↗
PubMedDate not supplied

Morphological, biochemical and molecular characterization of Herpetomonas samuelpessoai camargoi n. subsp., a trypanosomatid isolated from the flower of the squash Cucurbita moschata.

We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas-specific molecular markers based on spliced-leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed.

Animals↗
PubMed2004

Eradication of the melon fly, Bactrocera cucurbitae, in Japan: importance of behavior, ecology, genetics, and evolution.

The melon fly, Bactrocera cucurbitae, is a destructive insect of cucurbit and other fruits. It invaded the Southwestern Islands of Japan from 1919 to 1974. The sterile insect technique (SIT) was successfully applied from 1972 to 1993 to eradicate the melon fly. Technical research into SIT functions, such as suppression of density, mass-rearing, sterilization, shipment, release, evaluation of efficacy, and quality control of mass-reared insects, was conducted for this eradication project. Fundamental research into the dispersion, mating and oviposition behavior, population dynamics and estimation of density, eradication models, spatial distribution, genetics, and evolution of the melon fly was also undertaken and supported the success of the eradication project.

Animals↗
PubMed2006

Novel selective cytotoxicity of wild sarsaparilla rhizome extract.

Among six fractions, including total extract and fractions of hexane, ethyl acetate, butanol, water and boiling water extracted and separated from wild sarsaparilla rhizome, the hexane fraction (HRW) was the most effective in eliminating four different human cancer cell lines with cellular viability less than 6.8%. HRW exhibited the highest potency against human leukaemia cells with an IC50 (concentration that inhibited the growth rate of cells by 50%) of 3.3 +/- 0.3 microg mL(-1), which was 17.6-fold smaller than that against normal human umbilical vein endothelial cells (IC50, 58.0 +/- 1.5 microg mL(-1)). For its rich natural resources, simple extraction procedure and high yield (3.2%), HRW has the potential to be developed as a selective anti-cancer nutraceutical or pharmaceutical natural health product with low side effects and high economical return.

Antineoplastic Agents, Phytogenic↗
PubMed1983

Amino-acid sequence of two trypsin isoinhibitors, ITD I and ITD III from squash seeds (Cucurbita maxima).

The amino-acid sequences of two trypsin isoinhibitors, ITD I and ITD III, from squash seeds (Cucurbita maxima) were determined. Both isoinhibitors contain 29 amino-acid residues, including 6 half cystine residues. They differ only by one amino acid. Lysine in position 9 of ITD III is substituted by glutamic acid in ITD I. Arginine in position 5 is present at the reactive site of both isoinhibitors. The previously published sequence of ITD III has been shown to be incorrect.

Amino Acid Sequence↗
PubMed1985

Purification and characterization of the trypsin inhibitor from Cucurbita pepo var. patissonina fruits.

Three trypsin inhibitor fractions were found in white bush fruits (Cucurbita pepo L. var. patissonina). One of them, CPPTI-fIII, was purified to homogeneity by means of affinity and ion exchange chromatography. It is a cysteine-poor protein with an approximate Mr of 21 000. The inhibitor contains arginine at position P1 of the reactive site and inhibits bovine trypsin, hog pancreatic kallikrein and subtilisin. This inhibitor differs from the inhibitors of white bush dormant seeds, CPPTI-I and CPPTI-II, in its amino-acid composition, molecular mass, amino-acid residue at position P1 of the reactive site and inhibition spectrum.

Amino Acids↗
PubMed1990

Serine proteinase from Cucurbita ficifolia seed; purification, properties, substrate specificity and action on native squash trypsin inhibitor (CMTI I).

A proteinase was purified from resting seeds of Cucurbita ficifolia by ammonium sulfate fractionation and successive chromatography on CM-cellulose, Sephacryl S-300 and TSK DEAE-2SW (HPLC) columns. Inhibition by DFP and PMSF suggests that the enzyme is a serine proteinase. The apparent molecular mass of this enzyme is ca. 77 kDa. The optimum activity for hydrolysis of casein and Suc-Ala-Ala-Pro-Phe-pNA is around pH 10.5. The following peptide bonds in the oxidized insulin B-chain were hydrolysed by the proteinase: Phe1-Val2, Asn3-Gln4, Gln4-His5, Cya7-Gly8, Glu13-Ala14, Ala14-Leu15, Cya19-Gly20, Pro28-Lys29 and Lys29-Ala30. The proteinase is more selective towards the native squash seed trypsin inhibitor (CMTI I) and primarily cuts off only its N-terminal arginine. The inhibitor devoided of the N-terminal arginine residue is still active against trypsin.

Amino Acid Sequence↗
PubMed2000

Pollen competition in a natural population of Cucurbita foetidissima (Cucurbitaceae).

The pollen competition hypothesis predicts that when the number of pollen grains deposited onto stigmas exceeds the number of ovules, selection can operate in the time frame between deposition and fertilization. Moreover, because of the overlap in gene expression between the two phases of the life cycle, selection on microgametophytes may alter the resulting sporophytic generation. The extent to which pollen competition occurs in nature has been unclear, because tests of the predictions of the pollen competition hypothesis have used cultivars and/or artificial growth conditions and hand-pollination techniques. In this study we used a wild species, Cucurbita foetidissima, in its natural habitat (southern New Mexico) to determine the amount and timing of the arrival of pollen onto stigmas, the relationship between pollen deposition and seed number, and the effects of the intensity of pollen competition on progeny vigor. We found that ∼900 pollen grains are necessary for full seed set and that a single visit by a pollinator results in the deposition of 653.0 ± 101.8 pollen grains. About 29% of the flowers receiving a single pollinator visit had 900 or more pollen grains on its stigma. Moreover, within 2 h of anthesis, >4000 pollen grains were deposited onto a typical stigma, indicating that multiple pollinator visits must have occurred. Fruits produced by multiple visits had greater seed numbers (206 vs. 147) than fruits produced by a single visit. Finally, the progeny produced by multiple pollinator visits were more vigorous than those produced by single visits with respect to five measures of vegetative growth (MANCOVA, Wilks' lambda = 0.96, F(6,370) = 2.54, P < 0.02. These data demonstrate that conditions for pollen competition exist in nature and support the prediction that pollen competition enhances offspring vigor.

Journal Article↗
PubMed2000

Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity.

In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the pET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.

Arabidopsis↗
PubMed2005

[Genetic relationships among four Bactrocera Cucurbitae geographic populations in Yunan Province].

In this study, the partial section of mitochondrial cytochrome b in 23 individuals of four Bactrocera cucurbitae geographic populations (Banna, Ruili, Yuanjiang, and Hekou)in Yunnan Province was sequenced, and the phylogenetic trees of all haplotypes were constructed by means of N-J method, using B. dorsalis, B. correct and B. tau as the out-group species. Of the 426 bp mitochondrial cytochrome b DNA sequences obtained, A% + T% was about 65.0%, four nucleotide sites were substituted, and no base composition of the sequences was inserted and lost. Five haplotypes were identified in all the sequences, with four showing polymorphism, and one shared. The genetic characters of the four populations were analyzed by Fst value and gene flow through Mega software. The Fst value was 0.16667-0.20000 (P > 0.05), and the Nm value was 2.00 - 2.50. The N-J tree suggested that four haplotypes constructed three clades. All results showed that the genetic differences presented among the four populations, but with low degrees. The main reason causing the genetic differences among the four populations was supposed to be related to genetic isolation and ecological adaptation.

Animals↗