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Purification and structural analysis of an abundant thaumatin-like protein from ripe banana fruit.

The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-beta-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed.

Amino Acid Sequence↗

A biosynthetic intermediate of phytoalexins in banana fruits.

Phytoalexins of Musa balbisiana [BBB] cv Saba sa Hapon and cv Mundo fruits were compared with those of M. acuminata [AAA] cv Buñgulan fruits. Phytoalexins induced by wound and inoculation of Colletotrichum musae in these two cultivars were the same as those of Buñgulan. In the course of analysis of the phytoalexins, 2-phenyl-1,8-naphthalic anhydride, 2-(4'-methoxyphenyl)-1,8-naphthalic anhydride, and 1,2,3,4-tetrahydro-6,7-dihydroxy-1-(4'-hydroxycinnamyliden)naphthalen-2-one were found as new phytoalexins of banana fruits. This latter compound is probably a biosynthetic intermediate of phenylphenalenones in banana fruits.

Journal Article↗

Medicinal plants used for dogs in Trinidad and Tobago.

This paper documents ethnoveterinary medicines used to treat dogs in Trinidad and Tobago. In 1995, a 4-stage process was used to conduct the research and document the ethnoveterinary practices. Twenty-eight ethnoveterinary respondents were identified using the school-essay method, which is a modified rapid rural appraisal (RRA) technique. Semi-structured interviews were held with these respondents as well as with 30 veterinarians, 27 extension officers and 19 animal-health assistants and/or agricultural officers, and the seven key respondents that they identified. The final step involved hosting four participatory workshops with 55 of the respondents interviewed to discuss the ethnoveterinary data generated from the interviews and to determine dosages for some of the plants mentioned. Supplementary interviews were conducted in 1997 and 1998. Seeds of Carica papaya, and leaves of Cassia alata, Azadirachta indica, Gossypium spp., Cajanus cajan and Chenopodium ambrosiodes are used as anthelmintics. The anthelmintics Gossypium spp. and Chenopodium ambrosiodes are the most frequently used species. Crescentia cujete pulp, Musa spp. stem exudate, the inside of the pods of Bixa orellana, leaves of Cordia curassavica and Eclipta alba plant tops are used for skin diseases. Musa spp. stem exudate, seeds of Manilkara zapota, Pouteria sapota and Mammea americana and leaves of Cordia curassavica, Scoparia dulcis and Nicotiana tabacum are used to control ectoparasites. Dogs are groomed with the leaves of Cordia curassavica, Bambusa vulgaris and Scoparia dulcis. Psidium guajava buds and leaves and the bark of Anacardium occidentale are used for diarrhoea. Owners attempt to achieve milk let-down with a decoction of the leaves of Stachytarpheta jamaicensis. The plant uses parallel those practised in human folk medicine in other Caribbean countries and in other tropical countries.

Animals↗

In vitro efficacy of plant volatiles for inhibiting the growth of fruit and vegetable decay microorganisms.

The effects of acetaldehyde, benzaldehyde, cinnamaldehyde, ethanol, benzyl alcohol, nerolidol, 2-nonanone, beta-ionone, and ethyl formate vapors on the growth of Rhizopus stolonifer, Penicillium digitatum, Colletotrichum musae, Erwinia carotovora, and Pseudomonas aeruginosa on agar medium were evaluated. The aldehydes were found to be the strongest growth inhibitors and the most lethal to the fungal spores and mycelia and bacterial cells. The average minimum inhibitory concentrations (MICs) of aldehydes that were germicidal to decay microorganisms were 0.28, 0.49, and 0.88 mmol per Petri dish, for cinnamaldehyde, benzaldehyde, and acetaldehyde, respectively. Ethanol also inhibited growth completely, but the MIC, which was 14.6 mmol per Petri dish, was significantly higher than those of the aldehydes. Ethanol can be considered germistatic because the alcohol does not inhibit germination of spores completely; it completely controlled only mycelial growth. The ketones tended to be effective only on P. digitatum and C. musae, whereas ethyl formate was not effective except on P. digitatum. The concentration of a volatile compound in the headspace of the Petri dish and its diffusion into the medium largely determined its efficacy against decay microorganisms.

Agar↗

Colonization pattern of aquatic hyphomycetes on leaf packs in subtropical stream.

Steralized leaf pieces of eight plants (Eucalyptus rostrata, Phoenix dactylifera, Phragmites australis, Musa nana, Salix subserrata, Cyperus alopecuroides, Ricinus communis and Eichhornia crassipes) were submerged in the Nile stream. Thirty-nine species of aquatic hyphomycetes were colonized on the plant leaves. Eucalyptus was the best substratum (30 species) for colonization by aquatic hyphomycetes. Phoenix (14 species), Phragmites (11 species), Salix (9 species), Musa (8 species), Ricinus and Cyperus (6 species) were also good substrata for aquatic hyphomycetes colonization. Echhornia (aquatic plant) was not suitable for aquatic hyphomycetes colonization. Alatospora acuminata, Triscelophorus monosporus and Tetracladium marchalianum were found to be the major colonizers on all leaf materials. Temperature and dissolved oxygen were the highest physico-chemical parameters affecting the aquatic hyphomycetes colonization. These results indicate that aquatic hyphomycetes have been shown to be successful colonizers on plants leaf in river Nile in Egypt as subtropical region.

Journal Article↗

Bananas--physiology and biochemistry of storage and ripening for optimum quality.

Bananas (Musa spp.) are a major food crop of the humid tropics, and although edible cultivars are diverse and numerous, most of our knowledge of the physiology and biochemistry of these fruits relates to a few dessert cultivars of the AAA type, mainly of the Cavendish subgroup, which dominate the export trade between tropical and temperate zones. The preclimacteric period of banana fruits after harvest determines their transportability, and its duration is very sensitive to changes in fruit maturity, storage temperature, ethylene concentration, and other factors; progress in measurement and resolution of each of these effects is described. Changes in composition of the ripening fruits, especially in the development of flavor volatiles, are reviewed. Progress in understanding the integration of the biochemical changes controlling ripening in banana fruits is discussed. Recent work on storage, ripening, and factors relating to sensory assessment of fruit quality is discussed for cultivars of Musa types not used in major export trades.

Amino Acids↗

Distinction between wild and cultivated enset (Ensete ventricosum) gene pools in Ethiopia using RAPD markers.

In southwest Ethiopia, the cultivation area of Ensete ventricosum (enset) overlaps with the natural distribution area of this species. Analyses of genetic diversity were undertaken using RAPD to provide information for conservation strategies as well as evidence of possible gene flow between the different gene pools, which can be of interest for future improvement of cultivated enset. The extent of RAPD variation in wild enset was investigated in 5 populations in the Bonga area (Kefficho administrative region) and 9 cultivated clones. Comparisons were also made with some Musa samples of potential relevance for crop improvement. Nine oligonucleotide primers amplified 72 polymorphic loci. Population differentiation was estimated with the Shannon index (G'(ST)=0.10), Nei's G(ST) (0.12) and AMOVA (Phi(ST)=0.12), and appears to be relatively low when compared with outbreeding, perennial species in general. Cluster analysis (UPGMA) and principal component analysis (PCA) similarly indicated low population differentiation, and also demonstrated that cultivated clones essentially clustered distinctly from wild enset samples, suggesting that the present-day cultivated enset clones have been introduced to domestication from a limited number of wild progenitors. In addition, subsequent gene flow between wild and cultivated enset may have been prohibited by differences between modes of propagation and harvesting time; cultivated enset is propagated vegetatively through sucker production and the plant is generally harvested before maturity or flower set, thereby hindering pollination by wild enset or vice versa. A significant correlation was not found between genetic and geographical distances. The relatively high total RAPD diversity suggests that wild enset populations in the Bonga area harbour genetic variability which could potentially act as a source for useful or rare genes in the improvement of cultivated enset. As expected, E. ventricosum was clearly differentiated from the analysed Musa samples, that clustered in accordance with the present morphology- and molecular marker-based taxonomy of the genus.

DNA, Plant↗

Effects of intraventricular catecholamines on luteinizing hormone release in ovariectomized-steroid-primed rats.

The effects of intraventricular norepinephrine (NE) and dopamine (DA) were studied in the awake, freely behaving rat. In long-term ovariectomized, estrogen-progesterone-primed (OVE E2-P) animals, blood samples were taken via indwelling intra-atrial catheters before and after intraventricular infusion of either pH-adjusted saline, NE )5 mug, 15 mug, 20 mug), or DA (4 mug, 15 mug), and plasma LH was measured by radioimmunoassay. Under urethane anesthesia, records were made of the effects of intraventricular saline and NE on the electrical activity of the arcuate nucleus in the form of multi-unit spike activity. In unanesthetized animals, intraventricular NE caused marked changes in behavior. The typical response consisted of three phases: generalized activation (5-7 min), feeding (5-15 min), and sleep 1-2 h). DA exerted similar behavioral effects but without the marked sleep phase characteristic of the NE response. The effects of the catecholamines on LH output were significant increases in plasma LH levels for all NE doses tested (5 mug, p less than .025; 15 mug, p less than .05; 20 mug, p less than .005), while DA had no effect. The dynamics of the LH response to NEwere similar at all dosage levels, and the increase caused by 20 mug NE was found to be essentially equal to that induced by a quick intravenous infusion of 1.25 ng LHRH. Arcuate nucleus multi-unit spike activity (MUSA) showed a clear response to intraventricular NE at a dosage capable of stimulating the release of LH. In every case, the initial effect was a decrease in spike activity. These results, considered in relation to previous findings, suggest that NE may be stimulatory to neurons secreting LH-releasing hormone (LHRH). The decrease in arcuate nucleus MUSA in response to NE implies that certain elements of this nucleus are inhibited during LH release, perhaps the dopaminergic tuberoinfundibular neurons.

Animals↗

Triterpene phytoalexins from strawberry fruit.

Strawberry cv. Houkouwase is resistant to infection by Colletotrichum fragariae. The formation of antifungal compounds was observed in unripe fruit which had been wounded and inoculated with conidia of C. musae. Three antifungal compounds were isolated and identified as euscaphic acid, tormentic acid and myrianthic acid. Myrianthic acid inhibited the growth of C. musae at 3 microg, and euscaphic and tormentic acids showed inhibitory effects at 100 microg. A quantitative analysis of their contents showed that the triterpenes increased in wounded fruit, and in wounded and inoculated fruit, but not in non-treated fruit. These findings indicate that unripe fruit of Houkouwase produced the triterpenes as phytoalexins. The triterpene phytoalexins seem to be involved in the resistance of strawberry to the fungus.

Anti-Infective Agents↗

Changes in the content and biosynthesis of phytoalexins in banana fruit.

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-(13)C] and [2-(13)C]cinnamic acids, and [2-(13)C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4'-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.

Chromatography, High Pressure Liquid↗

[The study of the major sarcosaphagous flies in Hohhot].

OBJECTIVE: In order to determine the major species of sarcosaphagous flies and their regular activity on carcass in Hohhot district. METHODS: Six rabbits were killed and placed outdoors at different time from July to October in Hohhot district. Some species of sarcosaphagous flies that appeared the cadavers were observed and identified. RESULTS: It showed that there are 10 main spieces belonging to 3 families and 8 genera were from Diptera, including Musa domestica domestica Linnaeus, Musa domestica vicina Maequart, Ophyra capensis (Wiedemann), Hydrotaea armipes (Fall.), Muscina stabulans (Fall.) from Museidae; Lucilia sericata (Meigen), Chrysomya megacephala (Fabricius), Lucilia cuprina (Wiedemann) from Calliphoridae; Boettcherisca peregrina (Robineau-Desvoiy), Parascarcophaga crassipalpis (Maequart) from Sacrophagidae. Besides, it showed obvious regulations that different species of sarcosaphagous flies appeared on carcass at different postmortem interval and the first part of carcass that sarcosaphagous flies appeared on. CONCLUSION: It may be useful for estimating postmortem interval in Hohhot district.

Animals↗

[On the similarity of antigen between torulopsis and salmonella. 2. (communication) (author's transl)].

The immune sera for Torulopsis castellii, T. ernobii, T. humilis, T. insectalens, T. musae, T. pintolopesii and T. silvatica agglutinated Salmonella cholerae suis (6,7). The immune serum for S. cholerae suis agglutinated Torulopsis castellii, T. ernobii, T. humilis, T. insectalens, T. musae, T. pintolopesii and T. silvatica. Absorption and agglutination cross test demonstrated common antigen factors in the tested Torulopsis species and in S. cholerae suis (6,7).

Agglutination Tests↗

Activity and expression of banana starch phosphorylases during fruit development and ripening.

Two main forms of starch phosphorylase (EC 2.4.1.1) were identified and purified from banana (Musa acuminata Colla. cv. Nanicão) fruit. One of them, designated phosphorylase I, had a native molecular weight of 155 kDa and subunit of 90 kDa, a high affinity towards branched glucans and an isoelectric point around 5.0. The other, phosphorylase II, eluted at a higher salt concentration from the anion exchanger, had a low affinity towards branched glucans, a native molecular weight of 290 kDa and subunit of 112 kDa. Kinetic studies showed that both forms had typical hyperbolic curves for orthophosphate (Pi) and glucose-1-phosphate, and that they could not react with substrates with a blocked reducing end or alpha-1,6 glucosidic bonds. Antibodies prepared against the purified type-II form and cross-reacting with the type-I form showed that there was an increase in protein content during development and ripening of the fruit. The changes in protein level were parallel to those of phosphorylase activity, in both the phosphorolytic and synthetic directions. Considering the kinetics, indicating that starch phosphorylases are not under allosteric control, it can be argued that protein synthesis makes a contribution to regulating phosphorylase activity in banana fruit and that hormones, like gibberellic acid and indole-3-acetic acid, may play a regulating role. For the first time, starch phosphorylases isoforms were detected as starch-granule-associated proteins by immunostaining of SDS-PAGE gels.

Fruit↗

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit.

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.

Adenosine Triphosphate↗

Molecular cloning and characterisation of banana fruit polyphenol oxidase.

Polyphenol oxidase (PPO; EC 1.10.3.2) is the enzyme thought to be responsible for browning in banana [Musa cavendishii (AAA group, Cavendish subgroup) cv. Williams] fruit. Banana flesh was high in PPO activity throughout growth and ripening. Peel showed high levels of activity early in development but activity declined until ripening started and then remained constant. PPO activity in fruit was not substantially induced after wounding or treatment with 5-methyl jasmonate. Banana flowers and unexpanded leaf roll had high PPO activities with lower activities observed in mature leaves, roots and stem. Four different PPO cDNA clones were amplified from banana fruit (BPO1, BPO11, BPO34 and BPO35). Full-length cDNA and genomic clones were isolated for the most abundant sequence (BPO1) and the genomic clone was found to contain an 85-bp intron. Introns have not been previously found in PPO genes. Northern analysis revealed the presence of BPO1 mRNA in banana flesh early in development but little BPO1 mRNA was detected at the same stage in banana peel. BPO11 transcript was only detected in very young flesh and there was no detectable expression of BPO34 or BPO35 in developing fruit samples. PPO transcripts were also low throughout ripening in both flesh and peel. BPO1 transcripts were readily detected in flowers, stem, roots and leaf roll samples but were not detected in mature leaves. BPO11 showed a similar pattern of expression to BPO1 in these tissues but transcript levels were much lower. BPO34 and BPO35 mRNAs were only detected at a low level in flowers and roots and BPO34 transcript was detected in mature leaves, the only clone to do so. The results suggest that browning of banana fruit during ripening results from release of pre-existing PPO enzyme, which is synthesised very early in fruit development.

Acetates↗

Purification and characterization of banana fruit acid phosphatase.

An acid phosphatase (APase, EC 3.1.3.2) from ripened banana (Musa cavendishii L. cv. Cavendish) fruit has been purified 1,876-fold to electrophoretic homogeneity and a final p-nitrophenylphosphate (pNPP)-hydrolyzing specific activity of 745 micromol Pi produced (mg protein)(-1) min(-1). Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with APase activity. SDS-PAGE and analytical gel filtration demonstrated that the purified enzyme exists as a 40-kDa monomer. That the enzyme is glycosylated was indicated by its tight absorption to Concanavalin A-Sepharose. Banana APase was relatively heat stable, displayed a symmetrical pH/activity profile with maximal activity at pH 5.8, and was activated 180% and 150% by 5 mM Mn2+ and Mg2+, respectively. The enzyme exhibited a broad substrate selectivity, with maximal specificity constants (Vmax/Km) obtained with pNPP, phosphoenolpyruvate, phenyl phosphate, and O-phospho-L-tyrosine. Potent inhibition by Pi, molybdate, vanadate, arsenate, and Zn2+ was observed. Putative metabolic functions of the APase are discussed in relation to maintaining significant Pi mobility during banana fruit ripening.

Acid Phosphatase↗

Molecular characterization of banana virus X (BVX), a novel member of the Flexiviridae family.

A novel virus was identified in banana (Musa spp). Analysis of the last 2917 nucleotides of its positive strand genomic RNA showed five open reading frames corresponding, from 5' to 3', to a truncated ORF coding for a replication-associated protein, three ORFs coding for a movement-associated triple gene block (TGB) and a capsid protein (CP) gene. This genome organization is similar to that of some members of the Flexiviridae family such as potexviruses and foveaviruses. This virus was named Banana virus X (BVX). Comparative sequence analysis showed that BVX is only distantly related to other members of the Flexiviridae family, in which it appears to define a new genus. BVX produces defective RNAs derived from its genomic RNA by non-homologous recombination. Three distinct pairs of donor/acceptor recombination sites involving short direct nucleotide repeats were characterized, accounting for deletions of 1268, 1358 and 1503 nucleotides. Contrary to the situation encountered for Potexviruses, these recombination sites are located within the TGB1 and CP genes and result in a truncated TGB1 protein.

Capsid Proteins↗

Composition, digestibility and application in breadmaking of banana flour.

Banana flour (BF) was obtained from unripe banana (Musa paradisiacal L.) and characterized in its chemical composition. Experimental bread was formulated with BF flour and the product was studied regarding chemical composition, available starch (AS), resistant starch (RS) and rate of starch digestion in vitro. The chemical composition of BF showed that total starch (73.36%) and dietary fiber (14.52%) were the highest constituents. Of the total starch, available starch was 56.29% and resistant starch 17.50%. BF bread had higher protein and total starch content than control bread, but the first had higher lipid amount. Appreciable differences were found in available, resistant starch and indigestible fraction between the bread studied, since BF bread showed higher resistant starch and indigestible fraction content. HI-based predicted glycemic index for the BF bread was 65.08%, which was significantly lower than control bread (81.88%), suggesting a "slow carbohydrate" feature for the BF-based goods. Results revealed BF as a potential ingredient for bakery products containing slowly digestible carbohydrates.

Biological Availability↗