Adult and larva of Aedes (Finlaya) sorsogonensis Bañez, 1963. Comparison with Aedes (Finlaya) ananae Knight and Laffoon, 1946 and Aedes (Finlaya) poecilus (Theobald), 1903.
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[Effect of the addition of tropical fruits--pineapple (Ananas comosus) and papaya (Carica papaya)--on the production of biological silage from fish].
Microbial Fish Silage was produced from under-utilized fish mixed with juice and waste fruits (pineapple and papaya) at 35 degrees C. Six different products were elaborated as following: A: fish muscle with pineapple juice; B: fish muscle with fruit wastes; C: gutted fish with pineapple juice; D: gutted fish with fruit wastes; E: whole fish with pineapple juice; F: whole fish with fruit wastes. Process development was evaluated by measuring: pH, acidity, non-protein nitrogen, consistency and exudate liquid. Results indicated a slow decrease in pH value and production of acidity during 20 storage days. The addition of fruits to silage did not have any effect on these values. Silage liquefaction or hydrolysis was related to the following parameters: consistency, non-protein nitrogen and exudate liquid. The addition of fruits was related to silage liquefaction or hydrolysis, and it was measured by the consistency. Exudate liquid and non-protein nitrogen increased during storage time. However silage made from whole fish showed highest values in those parameters than other processing conditions. This results suggested that hydrolysis involve first enzymes from guts and second enzymes from muscle and head. Pineapple juice did not contribute to hydrolysis process. Mainly sensory changes in the silage occurs during first 24 hours and they were related to chemical changes. Proximal analysis did not change during silage process and microbial counts indicated the freshness of raw material used i this study.
Animals↗
Biological control of an insect pest by gut-colonizing Enterobacter cloacae transformed with ice nucleation gene.
The ice nucleation (IN) gene inaA of epiphytic Erwinia (Pantoea) ananas IN10 was transformed into Enterobacter cloacae WBMH-3-CMr originated from the faeces of silkworms. The transformant designated as Ent. cloacae WBMH-3-CMr(pICE6S13) exhibited IN activity, unlike the parent strain. The transgenic strain was ingested by mulberry pyralid larvae, fed on detached mulberry leaves, and the supercooling capacity and cold hardiness of these larvae were examined. The mean supercooling point (SCP) of the larvae ingesting the transgenic strain was - 3.3 degrees C, 8 degrees C higher than that of larvae treated with distilled water (control) and 1.5 C higher than an ice nucleation active (INA) strain of Erw. ananas. The SCPs of the larvae were stably maintained over the 9 d after ingestion. The maintenance of these high SCPs was due to transgenic Ent. cloacae having a more stable and efficient gut colonization than Erw. ananas, which is identified by the distribution of a narrower range of SCPs (-2 to -5 degrees C) in larvae treated with the transgenic stain. Furthermore, most of the larvae ingesting the transgenic strain froze and died when they were exposed to cold conditions of -5 degrees C for 18 h, 3 or 7 d after ingestion. In contrast, most of the larvae ingesting no bacterium did not die under similar conditions. On the other hand, the growth ability of Ent. cloacae WBMH-3-CMr on mulberry leaves tended to be lower than that of epiphytic Erw. ananas, as assayed by pot tests. These findings would expand the possibility of biological control using INA bacteria since Ent. cloacae would harbour a broader host (insect) range for gut colonization and a smaller affinity to plants to benefit from prevention of plant frost injury.
Animals↗
Fructose 2,6-bisphosphate, carbohydrate partitioning, and crassulacean Acid metabolism.
Fructose 2,6-bisphosphate (F 2,6-P(2)) was detected in the CAM species, Ananas comosus and Bryophyllum tubiflorum, and in C(3)- and CAM-Mesembryanthemum crystallinum. In both Mesembryanthemum tissues, F 2,6-P(2) was located outside the chloroplast. The levels of F 2,6-P(2), malate, starch, or soluble sugars were measured during various periods during the day-night cycle in the leaves of Ananas, a species which stores carbohydrate in an extrachloroplastic compartment, and in Bryophyllum, a species which stores carbon as starch in the chloroplast. In both species, the levels of F 2,6-P(2) were correlated with the stages of the day-night CAM cycle. Immediately following the dark-light transition the F 2,6-P(2) levels exhibited a rapid transient increase followed by a decrease. F 2,6-P(2) reached a daily minimum soon after the onset of deacidification and remained low until the malic acid pools approached their daily minima; the levels of F 2,6-P(2) then began a slow increase which accelerated during the period of afternoon CO(2) uptake. Immediately following the light-dark transition F 2,6-P(2) levels fluctuated. The levels were usually low after the fluctuations had ceased. The pools then increased as the rate of malate synthesis increased, remained at relatively constant high levels when the rates of malate synthesis were constant, and decreased as malate synthesis decreased towards the end of the dark period. The absolute levels of F 2,6-P(2) were always higher in Ananas than in Bryophyllum. The ratios of the activity of pyrophosphate fructose-6-phosphate l-phosphotransferase to cytoplasmic fructose 1,6-bisphosphatase and to phosphofructokinase were also far higher in Ananas than in Bryophyllum or in C(3)- or CAM-Mesembryanthemum.
Journal Article↗
Detection of variation of the R-domain structure of ice nucleation genes in Erwinia herbicola-group bacteria by PCR-RFLP analysis.
The structure of ice nucleation (IN) genes was compared among 20 strains of Erwinia herbicola-group bacterium of plant- and insect-origin including E. herbicola M1 (IceE) and E. ananas IN10 (inaA) that had been previously reported. When the DNAs of N-domain or C-domain were amplified, PCR products with similar size were obtained in all strains, while the size of the PCR products from the whole genes containing the R domain varied remarkably within a range of 3.8 kb to 4.4 kb. RFLP analysis of the IN genes revealed that the size of the R-domains were varied within the region from the PvuII site to DraI site, and 20 IN genes were classified into 12 groups. Furthermore, all the strains identified as E. ananas based on six bacteriological properties were different from those of E. herbicola. These results suggest that the IN genes may be distributed only in E. ananas strains among "herbicola group bacteria."
Animals↗
Sulfur dioxide effects on plants exhibiting Crassulacean Acid Metabolism.
The effects of SO(2) on species exhibiting Crassulacean Acid Metabolism (CAM) were determined with short term-high concentration 'acute' greenhouse exposures (0.6 to 3.0 microl liter(-1) (ppm) SO(2) for 2 and 8 h), and long term-low concentration 'chronic' field exposures (0.35 to 0.90 microl liter(-1) SO(2) for 32 to 79 h periodically over 7 to 13 days). In the acute greenhouse exposures, visible injury was observed on Opuntia basilaris Engelm. & Bigel., exposed to 2.0 microl liter(-1) SO(2), but no injury was observed on Ananas comosus (L.) Merr., Bryophyllum blossfeldiana Poelln., Bryophyllum pinnata (Lam.) Pers., or Bryophyllum tubiflora (Harv.) Hamet, exposed to up to 2.8 microl liter(-1) SO(2) for 8 h. Stomatal conductance during the exposures averaged 0.067+/-0.021mol(-2)s(-1) for Opuntia basilaris, 0.029+/-0.008mol(-2)s(-1) for Ananas comosus, and 0.029+/-0.008mol m(-2)s(-1) for Bryophyllum pinnata. Opuntia basilaris was injured early during the day, but not at night; with the injury appearing as a white necrotic banding across just fully expanded pads. Moderately injured pads would regreen beginning 1 to 2 weeks after exposure. In chronic field exposures, no visible injury from SO(2) was observed on Opuntia basilaris, Dudleya arizonica Rose or Agave deserti Engelm. plants, grown either with supplemental irrigation or natural rainfall. In addition, in the field SO(2) had no effect on CO(2) uptake, total sulfur content, transpiration, or tissue acidity in either the light or the dark, or in irrigated vs natural rainfall plots.
Journal Article↗
Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae.
The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at -2.5 degrees C, significantly higher than their parent strains (active at approx -10 degrees C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were -3 to -4 degrees C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of -7 degrees C, and more than 95% died after 12 h at -5 degrees C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields.
Agriculture↗
Distribution and density of mosquitoes in two endemic areas for bancroftian filariasis in Sorsogon, Philippines.
Mosquito density in Rangas where abaca is in abundance is much higher, almost twice, than that of Putiao where abaca is absent. The adult density of Aedes poecilus over Aedes ananae in the two areas combined is 3 to 4 times whereas the larval density of the former is much lower than Aedes ananae. The banana axils is a favorite breeding place for Aedes poecilus but may also utilize the abaca axils. This finding is very favorable in the transmission of bancroftian filariasis because this species of banana is planted around houses even closer to human dwelling than the abaca plants. Aedes poecilus being more anthropophylic than zoophylic can be domesticated as Culex quinquefasciatus and Aedes aegypti which again is a factor in favor of ideal transmission of the disease. The fact that bananas are planted around houses both in villages and towns, transmission of the disease could easily occur in both places, which should be borne in mind when planning a control program. The number of dissected mosquitoes is too small to be of significance in vector determination.
Animals↗
Purification and characterization of a pineapple crown leaf thiol protease.
A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.
Ananas↗
Day-night changes of energy-rich compounds in crassulacean acid metabolism (CAM) species utilizing hexose and starch.
BACKGROUND AND AIMS: Plants with crassulacean acid metabolism (CAM) can be divided into two groups according to the major carbohydrates used for malic acid synthesis, either polysaccharide (starch) or monosaccharide (hexose). This is related to the mechanism and affects energy metabolism in the two groups. In Kalanchoë pinnata and K. daigremontiana, which utilize starch, ATP-dependent phosphofructokinase (tonoplast inorganic pyrophosphatase) activity is greater than inorganic pyrophosphate-dependent phosphofructokinase (tonoplast adenosine triphosphatase) activity, but the reverse is the case in pineapple (Ananas comosus) utilizing hexose. To test the hypothesis that the energy metabolism of the two groups differs, day-night changes in the contents of ATP, ADP, AMP, inorganic phosphate (Pi), phosphoenolpyruvate (PEP) and inorganic pyrophosphate (PPi) in K. pinnata and K. daigremontiana leaves and in pineapple chlorenchyma were analysed. METHODS: The contents of energy-rich compounds were measured spectrophotometrically in extracts of tissue sampled in the light and dark, using potted plants, kept for 15 d before the experiments in a growth chamber. KEY RESULTS: In the three species, ATP content and adenylate energy charge (AEC) increased in the dark and decreased in the light, in contrast to ADP and AMP. Changes in ATP and AEC were greater in Kalanchoë leaves than in pineapple chlorenchyma. PPi content in the three species increased in the dark, but on illumination it decreased rapidly and substantially, remaining little changed through the rest of the light period. Pi content of Kalanchoë leaves did not change between dark and light, whereas Pi in pineapple chlorenchyma increased in the dark and decreased in the light, and the changes were far greater than in Kalanchoë leaves. Light-dark changes in PEP content in the three species were similar. CONCLUSIONS: These results corroborate our hypothesis that day-night changes in the contents of energy-rich compounds differ between CAM species and are related to the carbohydrate used for malic acid synthesis.
Adenosine Diphosphate↗
Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form.
We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.
Ananas↗
Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem.
A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.
Amino Acid Sequence↗
Purification and characterization of macrodontain I, a cysteine peptidase from unripe fruits of Pseudananas macrodontes (Morr.) harms (Bromeliaceae).
A new papain-like cysteine peptidase isolated from fruits of Pseudananas macrodontes (Morr.) Harms, a species closely related to pineapple (Ananas comosus L.), has been purified and characterized. The enzyme, named macrodontain I, is the main proteolytic component present in fruit extracts and was purified by acetone fractionation followed by anion-exchange chromatography. Separation was improved by selecting both an adequate pH value and a narrow saline gradient. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1-8.5. Homogeneity of the enzyme was confirmed by bidimensional electrophoresis and mass spectroscopy (MS). Molecular mass of the enzyme was 23,459 (MS) and its isoelectric point was 6.1. The alanine, glutamine, and tyrosine derivatives were strongly preferred when the enzyme was assayed on N-alpha-CBZ-l-amino acid p-nitrophenyl esters. The N-terminal sequence of macrodontain (by comparison with the N-terminus of 30 plant proteases with more than 50% homology) showed a great deal of sequence similarity to the other pineapple-stem-derived cysteine endopeptidases, being 85.7, 85. 2, and 77.8% identical to comosain, stem bromelain, and ananain, respectively. It seems clear that the Bromeliaceae endopeptidases are more closely related to each other than to other members of the papain family, suggesting relatively recent divergence.
Amino Acid Sequence↗
Isolation and partial characterization of basic proteinases from stem bromelain.
Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substrate L-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine-->serine) and position 20 (asparagine-->glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0:2.0:1.0:2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. The pH optimum of F4 and F5 was between pH 4.0 and 4.5 and for F9 close to neutral pH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM, kcat 0.87 sec-1 and F5 (Km 2.42 mM, kcat 0.68 sec-1), and differed greatly from F9 (Km 0.40 mM, kcat 3.94 sec-1).
Amino Acid Sequence↗
Effect of pH, temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain.
The biological significance of the carbohydrate moiety of a glycoprotein has been a matter of much speculation. In the present work, we have chosen stem bromelain from Ananas comosus as a model to investigate the role of glycosylation of proteins. Stem bromelain is a thiol protease which contains a single hetero-oligosaccharide unit per molecule. Here, the deglycosylated form of the enzyme was obtained by periodate oxidation. The differences in the glycosylated and deglycosylated forms of the glycoprotein have been studied at various temperatures and pH values, using probes such as loss of enzyme activity and by the changes in fluorescence and circular dichroism spectra. Deglycosylated bromelain showed decreased enzyme activity and perturbed fluorescence and circular dichroism spectra. In addition to this, a comparative study of their activities in different organic solvents showed a marked decrease in case of deglycosylated form of the enzyme. It is thus concluded that glycosylation contributes towards the functional stability of glycoenzymes.
Alcohols↗
Serological difference between Erwinia herbicola strains of plant and human origin.
Fifty Erwinia herbicola isolates obtained from host plants were examined in an agglutination reaction with antiserum prepared against E. ananas (E. herbicola) strain CCM 2407 antigen of plant origin and with antiserum prepared against Enterobacter agglomerans strain CNCTC M 269 antigen of human origin. In tests with strain CCM 2407 antiserum, 56% isolates showed a positive reaction, while in tests with strain CNCTC M 269 antiserum only 14% isolates showed a positive reaction. Among E. herbicola isolates which showed a positive reaction with CCM 2407 antiserum 18% showed a positive reaction with the CNCTC M 269 antiserum too. Our results confirmed the serological heterogeneity of E. herbicola population. In spite of the difference in the origin of the two antigens used for the preparation of antisera (plant, human; Japan, Czech Republic) our results indicate that some of our E. herbicola strains and E. agglomerans strain CNCTC M 269 are serologically identical.
Agglutination Tests↗
Amino acid composition of food products used in the treatment of patients with disorders of the amino acid and protein metabolism.
The amino acid composition of food products frequently used in the diets of amino acid and protein disorders-including tryptophan- was estimated using ion-exchange column chromatography and high performance liquid chromatography. It includes fruits (different varieties of apples, pears, ananas, bananas, peach, strawberries, honey melon, water melon, kiwi, plums, grapes), vegetables (different varieties of potatoes, potato products, cauli-flower, broccoli, cabbages, spinach, olives, lettuce, cucumber, peas, mushrooms) and commercially available or home-made food products (meat broth, fine gravy paste, ketchup, liquid seasoning, soja sauce, different varieties of Chinese noodles, sausages for phenylketonuria patients), and different new fiber concentrates.
Amino Acid Metabolism, Inborn Errors↗
Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants.
The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed. </hea
Journal Article↗