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[Preclinical studies of cucurbita maxima (pumpkin seeds) a traditional intestinal antiparasitic in rural urban areas].

Experimental research was carried out at the Parasitology and Chemistry laboratories of the Jorge Basadre Grohmann National University, in Tacna. The process involved two phases: (1) determination of the minimum inhibitory concentration (MIC) of Cucurbita Maxima as an antiparasitic agent using canine tapeworms with an intestinal isolation of 5 to 6 hours, and (2) determination of the side-effects of Curbita Maxima on exposed albino rats. It was found that the MIC of 23 gr. of pumpkin seed in 100 ml. of distilled water can produce an antihelminthic effect. This concentration is equivalent to +/- 73 pumpkin seeds (x2 = 5.6, p<0.01). Macroscopically, alterations in helminthic motility are present at a dose of > 23 gr. There is a protheolithic effect with an average survival time of 38.4 minutes. Microscopically the mature proglottids present a destruction of the tegument involving the basal membrane. In the gravid proglottids there is egg destruction. These findings are accentuated when experimenting with Cucurbita Maxima in a concentration of 30 and 32 gr. Superficial non-erosive gastritis was found in weys rats after 4 hours of administering 9 gr/kg.

Animals↗

Organization and characterization of Cucurbita phloem lectin genes.

The phloem of pumpkin and squash contains a dimeric chitin-binding lectin called PP2 (phloem protein 2). We have isolated three genomic clones from pumpkin (Cucurbita maxima Duch.) that encoded PP2. One clone, lambda gPC13-1, contained two PP2 genes that were 99.8% identical over a region of 3055 nucleotides. This conserved region included 1922 bp of 5' non-coding sequence, 844 bp of protein coding sequence (including two introns), and 289 bp of 3' non-coding sequence. To examine the conservation of the phloem lectin within the genus Cucurbita, we analyzed nine different species for PP2, its mRNA, and the genes that encode PP2. DNA blot analysis indicated that each species contained genes that encoded PP2, however, there was considerable restriction fragment length polymorphism (RFLP) among the species. PP2 gene copy number reconstructions indicated that PP2 is encoded by a small gene family (two to eight genes). Although a high level of PP2 DNA polymorphism existed among species, a single mRNA (ca. 1 kb) was detected in each species. PP2, affinity-purified from the vascular exudate of each species, reacted with PP2-specific antibodies; five species contained a single PP2 polypeptide while four species contained two PP2 polypeptides.

Amino Acid Sequence↗

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo.

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequences on two cyanogen bromide fragments were determined. An oligonucleotide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3-5-day old seedling hypocotyls, in lambda ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539-1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7-15 per haploid genome).

Amino Acid Sequence↗

Differential homogenization and amplification of two satellite DNAs in the genus Cucurbita (Cucurbitaceae).

Two different satellite DNAs exist in the genus Cucurbita which are different with respect to repeat length (350 bp and 170 bp), array size, and sequence homogenization. Whereas the 350-bp satellite DNA is prominent and very homogeneous in all species investigated except for C. maxima and C. lundelliana, the 170-bp satellite is rather evenly distributed in all species. In C. maxima and C. lundelliana the 350-bp satellite is present only in small amounts, but detectable by the sensitive PCR method. These repeats are also very homogeneous, reflecting a silent stage of satellite DNA. In contrast, the 170-bp satellite DNA is intra- and interspecifically heterogeneous. It is striking that the species with no detectable amount of 350-bp satellite contain 170-bp satellite DNA clusters with the highest degree of homogeneity. The evolution of satellite DNA repeats within cultivated and wild species in the genus Cucurbita is elucidated using the sequence data of both satellite DNAs from all species investigated. The value of satellite DNA for phylogenetic analysis between closely related species is discussed.

Base Sequence↗

Variability of vitamin E content in pumpkin seeds (Cucurbita pepo L.).

Pumpkin (Cucurbita pepo L.) seed oil is a common salad oil which is produced in the southern parts of Austria, Slovenia and Hungary. It is dark green and has a high content of free fatty acids. Due to its colour, the oil cannot be used for cooking. The content of vitamin E, especially gamma-tocopherol, is very high. The oil content of the pumpkin seed is about 50%. The seed itself can be eaten. Therefore a pumpkin variety with high vitamin E content is desirable. The aim of this work was to find a variety of Cucurbita pepo which has a high oil yield and a high vitamin E content. A total of 100 breeding lines were tested for their tocopherol content. The tocopherols and tocotrienols are extracted with hexane and analysed by NP-HPLC/FLD with hexane/dioxan (96/4) as eluent, with fluorescence detection at 292/335 nm. The gamma-tocopherol content, which is about 5-10 times as much as that of alpha-tocopherol varies over a broad range (41-620 mg/kg dry pumpkin seeds). Beta- and delta-tocopherol are found at low levels.

Chromatography, High Pressure Liquid↗

Cell division and cell enlargement in isolated Cucurbita cotyledons grown in darkness and in light.

The spatial and temporal patterns of post-embryonal cell growth and cell division were characterised in excised cotyledons of vegetable marrow ( Cucurbita pepo L. var. giromontia Alef.) incubated in water. The concurrent roles of these two processes in cotyledon growth were determined using paradermal sections of the first palisade layer of developing cotyledons. Tissue specificity was observed in the pattern of cell division. The daughter cells derived from an initial cell, which had already differentiated before imbibition of the seeds, were tightly packed in a cluster, which enabled us to monitor cell division during early cotyledon development. Heterogeneity of cell size was recognised during the process of cell proliferation in the cluster, suggesting that cell division is uncoupled from control of cell size. There was significantly more cell division in the marginal part of the cotyledons than in other parts, suggesting high activity of the marginal meristem. Light enhanced cell and cotyledon enlargement, but had no effect on the number of divisions. This study elucidated the cellular basis of post-germinative Cucurbita cotyledon morphogenesis and development.

Journal Article↗

Two linear plasmids in mitochondria of Fusarium solani f. sp. cucurbitae.

Two linear plasmid-like DNAs designated pFSC1 (9.2 kbp) and pFSC2 (8.3 kbp) were found in an isolate of the plant pathogenic fungus Fusarium solani f. sp. cucurbitae race 1. The plasmids were maternally inherited and copurified with mitochondrial DNA obtained from a mitochondria-enriched cell fraction suggesting that they are located in mitochondria. The plasmids did not share extensive sequence similarity. No homology was detected between either plasmid and the nuclear or mitochondrial genome when cloned plasmids were used as probes in Southern hybridization analyses. The fungus was cured of plasmids by ethidium bromide treatment. Compared to the plasmid-containing isolate, plasmid-cured derivatives had reduced pathogenicity on a susceptible plant host, Cucurbita maxima "Pink Banana."

DNA, Fungal↗

Hypoglycemic action of Cucurbita ficifolia on Type 2 diabetic patients with moderately high blood glucose levels.

Ten Type 2 diabetic patients with moderately hyperglycemia, received a raw extract of Cucurbita ficifolia or potable water in a single dose of 4 ml/Kg body weight, in two different sessions at least separated by 1 week. Blood glucose levels were followed in both parts of the study by a period of 5 h. The patients had stopped their pharmacologic medication 24 h prior to each part of the study. As expected, no significant changes on blood glucose were observed after giving the vehicle; however, the oral administration of C. ficifolia was followed by a significant decrease in blood glucose levels, from 12.07+/-1.69 mM (217.2+/-30.4 mg/dl) to 9.42+/-1.96 mM (169.6+/-35.3 mg/dl) 3 h after and to 8.37+/-1.74 mM (150.8+/-31.3 mg/dl) 5 h after the extract administration. The hypoglycemic action of Cucurbita ficifolia agrees with its effects previously observed in laboratory animals. The results of this preliminary study do not give indications for the mechanism of this hypoglycemic effect.

Adult↗

Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

Amino Acid Sequence↗

Olfactory receptors in the melon fly Dacus cucurbitae and the oriental fruit fly Dacus dorsalis.

Male melon flies (Dacus cucurbitae) from a colony in Hawaii were evaluated for limit of response to the olfactory stimulant raspberry ketone and to more than 40 related molecules. The results were compared with the limits of response of oriental fruit flies (Dacus dorsalis) under identical conditions. The nature of the response of the two species to attractive compounds appeared to be identical with regard to orientation, searching, pulsating mouthparts, and compulsive feeding. However, there was very little overlap in the response of the two species to phenylpropanoid-type compounds. D. cucurbitae responded most strongly to p-hydroxyphenylpropanoids while D. dorsalis responded most strongly to 3,4-dimethoxyphenylpropanoids. The results are discussed in terms of antennal receptor site geometry and with regard to the coevolution of two major groups of Dacini with plant kairomones.

Journal Article↗

Evolution of diabroticite rootworm beetle (Chrysomelidae) receptors for Cucurbita blossom volatiles.

The diabroticite rootworm beetles coevolved with plants of the family Cucurbitaceae as demonstrated by their feeding dependence on the tetracyclic triterpenoid cucurbitacins. These beetles also exhibit strong attraction to phenylpropanoid volatile components of Cucurbita blossoms. A mixture of 1,2,4-trimethoxybenzene, indole, and (E)-cinnamaldehyde, all blossom components, is highly attractive to the several species of diabroticite cucumber beetles and corn rootworms and is considered a simplified Cucurbita blossom kairomone odor. The evolutionary divergence in antennal receptor complementarity is best understood by comparing the species-specific responses of several Diabrotica to structural analogues of (E)-cinnamaldehyde, the major attractant for Diabrotica undecimpunctata howardi. Cinnamyl alcohol is a strong attractant for Diabrotica barberi, and 4-methoxycinnamaldehyde is an exceptional attractant for Diabrotica virgifera. The very closely related species D. barberi and Diabrotica cristata are most strongly attracted to 4-methoxyphenethanol, which is unattractive to the other species studied.

Journal Article↗

The regulation of gelation of Phloem exudate from cucurbita fruit by dilution, glutathione, and glutathione reductase.

The average glutathione equivalent concentration in phloem exudate collected from squash fruit (Cucurbita moschata [Duchesne] Poir. var Butternut) and pumpkin fruit (Cucurbita pepo [L.] var Jack-o-lattern) was 1.02 and 0.60 millimolar, respectively. Glutathione reductase (EC 1.6.4.2) activity in phloem exudate from squash and pumpkin fruit averaged 0.48 and 1.74 micromole NADPH oxidized per minute per milliliter, respectively. Protein concentrations in fruit phloem exudates averaged 67 milligrams per milliliter for squash and 57 milligrams per milliliter for pumpkin. The phloem-specific P-proteins account for most of the protein content of exudate. Pure exudate from fruit does not gel for hours or days, but when diluted with neutral or alkaline aqueous solutions, exudate gels rapidly. Exudate solutions undergo biphasic pH changes with dilution. We suggest that P-protein undergoes conformational change upon dilution, exposing titratable groups and sulfhydryl residues. Oxidation of the latter forms the intermolecular disulfide bridges of the gel. The gelation of diluted exudate is regulated by factors (oxygen, pH, glutathione, NADPH) which affect the maintenance of reduced sulfhydryl residues and the activity of glutathione reductase. While these factors may also act in vivo to regulate redox conditions in phloem, their relationship to hypothetical sol/gel transitions or motile and nonmotile phases in the transport conduit is unknown.

Journal Article↗

Polytene chromosome maps of the melon fly Bactrocera cucurbitae (Diptera: Tephritidae).

Standard photographic maps of the polytene chromosomes are presented for the melon fly Bactrocera cucurbitae, a serious pest of fleshy fruits and vegetables. Five larval salivary gland polytene chromosomes (10 polytene arms) were isolated, and their characteristic features and landmarks have been recognized. Banding patterns of each of the polytene arms are presented, where variation in band intensity and puffs appear to reflect fundamental differences in chromosomes. The whole polytene genome has been typically mapped by dividing it into 100 sections and the subsections were lettered. The mitotic chromosomes of larval brain ganglia are also examined, five pairs of autosomes and an XX/XY sex chromosome pair. In addition, a heterochromatic mass corresponding to the sex chromosomes are observed in the polytene nuclei of salivary gland tissue. This investigation showed that B. cucurbitae has excellent cytological material for polytene chromosome analysis and proved to be very useful for obtaining more detailed genetic information on the pest's natural populations.

Animals↗

Movement of sterile male Bactrocera cucurbitae (Diptera: Tephritidae) in a Hawaiian agroecosystem.

The melon fly, Bactrocera cucurbitae Coquillett, invaded the Hawaiian Island chain in 1895. In 1999, a program sponsored by the USDA-ARS to control melon fly and other tephritid pests in Hawaii over a wide area was initiated on the islands of Hawaii, Maui, and Oahu. To control these flies in an areawide setting, understanding how flies move within the landscape is important. To explore the movement of this fly, we examined the movement of marked, male, sterile, laboratory-reared B. cucurbitae on the island of Hawaii in an agricultural setting. Two releases of dyed, sterile flies consisting of approximately 15,000 flies, were released 6 wk apart. Released flies were trapped back by using Moroccan traps baited with a male attractant. These two releases suggest that in the Hawaiian agricultural areas where the areawide control is being sought, melon flies do not move extensively when there are abundant larval host and adult roosting sites. Over the course of this study, only one fly made it the maximum distance that we could detect fly movement (approximately 2,000 m in 2 wk). From these data, it seems that the flies dispersed throughout the study area but then moved very little thereafter. This is very apparent in the second release where the recovery rate after the second week was still fairly high, suggesting that if there are plenty of host fields and roosting sites the flies are unlikely to move.

Agriculture↗

In vivo monitoring of chlorophyll fluorescence response to low-dose gamma-irradiation in pumpkin (cucurbita pepo) leaves.

The effect of gamma-irradiation on the in vivo-measured chlorophyll fluorescence in a pumpkin leaves (Cucurbita pepo) has been investigated. Plants were grown in the same environment, then divided into several groups and irradiated at ambient conditions at small dose levels (up to 13.4 Gy) with (60)Co gamma-rays. The post-irradiation effect on chlorophyll status in the leaves was examined by measuring chlorophyll fluorescence 2 days (48 h) after exposure. It is undoubtedly found that the value of fluorescence intensity ratio (FIR) at 690 nm and 735 nm (F(690)/F(735)) depends upon the ionizing radiation dose. Even with the smallest dose of 3.35 Gy, ionizing radiation notably altered the fluorescence spectra of leaves. The spectra difference was manifested by decrease of FIR due to changed chlorophyll luminescence, the possible reason for which could be increase of chlorophyll concentration during the recovery process of the plant. The potential implications of these results for plant physiological status monitoring, as well as for pollution detection and assessment, are discussed in brief.

Chlorophyll↗

Characterization of Hungarian isolates of zucchini yellow mosaic virus (ZYMV, potyvirus) transmitted by seeds of Cucurbita pepo var Styriaca.

Zucchini yellow mosaic virus (ZYMV) has emerged as an important pathogen of cucurbits within the last few years in Hungary. The Hungarian isolates show a high biological variability, have specific nucleotide and amino acid sequences in the N-terminal region of coat protein and form a distinct branch in the phylogenetic tree. The virus is spread very efficiently in the field by several aphid species in a non-persistent manner. It can be transmitted by seed in holl-less seeded oil pumpkin (Cucurbita pepo (L) var Styriaca), although at a very low rate. Three isolates from seed transmission assay experiments were chosen and their nucleotide sequences of coat proteins have been compared with the available CP sequences of ZYMV. According to the sequence analysis, the Hungarian isolates belong to the Central European branch in the phylogenetic tree and, together with the ZYMV isolates from Austria and Slovenia, share specific amino acids at positions 16, 17, 27 and 37 which are characteristic only to these isolates. The phylogenetic tree suggests the common origin of distantly distributed isolates which can be attributed to widespread seed transmission.

Amino Acid Sequence↗

Effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low-protein fed rats.

The effects of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of lactate dehydrogenase (LD), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) against carbon tetrachloride (CCl4)-induced acute liver injury in low-protein fed rats were investigated. A group of male Sprague-Dawley rats maintained on a low-protein diet for 5 days were divided into three subgroups. Two subgroups were injected with carbon tetrachloride and the other group with an equivalent amount of olive oil. Two hours after CCl4 intoxication one of the two subgroups was administered with pumpkin seed protein isolate. All three subgroups of rats were maintained on the low-protein diet for the duration of the investigation. Groups of rats from the different subgroups were killed at 24, 48 and 72 h after their respective treatments. After 5 days on the low-protein diet the activity levels of all four enzymes were significantly higher than their counterparts on a normal balanced diet. CCl4 intoxication resulted in significant increases in the activity levels of all four enzymes investigated. The administration of pumpkin seed protein isolate after CCl4 intoxication resulted in significantly reduced activity levels of all four enzymes. It is concluded that pumpkin seed protein isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition.

Alanine Transaminase↗

In Vitro antioxidative activity of pumpkin seed (Cucurbita pepo) protein isolate and its In Vivo effect on alanine transaminase and aspartate transaminase in acetaminophen-induced liver injury in low protein fed rats.

The antioxidative effects of pumpkin seed protein isolate (Cucurbita pepo) were investigated in vitro. The isolate exhibited about 80% radical scavenging activity, chelating activity of approximately 64% on Fe2+ ions and an inhibition of approximately 10% of xanthine oxidase. Subsequently the effects of the isolate on the plasma activity levels of alanine transaminase and aspartate transaminase against acetaminophen induced acute liver injury in low-protein fed male Sprague-Dawley rats were ascertained. The rats were maintained on a low-protein diet for 5 days and divided into three subgroups. Two subgroups were injected with acetaminophen and the other with an equivalent amount of polyethylene glycol 400. Two hours after intoxication one of the two subgroups was administered with the protein isolate. Rats from the different subgroups were killed at 24, 48 and 72 h after treatment. After 5 days on the low-protein diet the activity levels of the enzymes were significantly higher than their counterparts on a normal balanced diet. The administration of protein isolate after acetaminophen intoxication resulted in significantly reduced activity levels. It is concluded that the protein isolate has promising antioxidative properties. Furthermore, the isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition and acetaminophen intoxication.

Alanine Transaminase↗