Carica papaya seed macerate as inhibitor of conjugative R plasmid transfer from Salmonella typhimurium to Escherichia coli in vitro and in the digestive tract of gnotobiotic mice.
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In this study, the effect of Carica papaya seed macerate on conjugal R plasmid transfer from Salmonella typhimurium to Escherichia coli was investigated in vitro and in the digestive tract of gnotobiotic mice. Twenty-five micrograms per milliliter and 430 mg (administered intragastrically twice a day) of papaya seed macerate concentrations were used during conjugation for in vitro and in vivo assays, respectively. High frequency of conjugation inhibition by macerate was observed for both in vitro and in vivo experiments, independently of bacterial growth and mating conditions. Papaya seed macerate caused a reduction of the transconjugant population ranging from 71% to about 100%. There was no lethal effect of the seed macerate on donor or recipient cells in the concentrations used. Once the mechanisms and magnitude of resistance gene transfer are clearly understood, strategies to reduce or minimize the dissemination of these genes could be relevant. The data here obtained show a clinical potential use of papaya seed macerate on this transfer. (+info)
Five ripening-related ACC synthase cDNA isoforms were cloned from 80% ripe papaya cv. 'Sinta' by reverse transcription-PCR using gene-specific primers. Clone 2 had the longest transcript and contained all common exons and three alternative exons. Clones 3 and 4 contained common exons and one alternative exon each, while clone 1, the most common transcript, contained only the common exons. Clone 5 could be due to cloning artifacts and might not be a unique cDNA fragment. Thus, there are only four isoforms of ACC synthase mRNA. Southern blot analysis indicates that all five clones came from only one gene existing as a single copy in the 'Sinta' papaya genome. Multiple sequence alignment indicates that the four isoforms arise from a single gene, possibly through alternative splicing mechanisms. All the putative alternative exons were present at the 5'-end of the gene comprising the N-terminal region of the protein. 'Sinta' ACC synthase cDNAs were of the capacs 1 type and are most closely related to a 1.4 kb capacs 1-type DNA (AJ277160) from Eksotika papaya. No capacs 2-type cDNAs were cloned from 'Sinta' by RT-PCR. This is the first report of possible alternative splicing mechanism in ripening-related ACC synthase genes in hybrid papaya, possibly to modulate or fine-tune gene expression relevant to fruit ripening. (+info)
Cloning and characterization of a FLORICAULA/LEAFY ortholog, PFL, in polygamous papaya.
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The homologous genes Floricaula (FLO) in Antirrhinum and LEAFY (LFY) in Arabidopsis are known to regulate the initiation of flowering in these two distantly related plant species. These genes are necessary also for the expression of downstream genes that control floral organ identity. We used Arabidopsis LFY cDNA as a probe to clone and sequence a papaya ortholog of LFY, PFL. It encodes a protein that shares 61% identity with the Arabidopsis LFY gene and 71% identity with the LFY homologs of the two woody tree species: California sycamore (Platanus racemosa) and black cottonwood (Populus trichocarpa). Despite the high sequence similarity within two conserved regions, the N-terminal proline-rich motif in papaya PFL differs from other members in the family. This difference may not affect the gene function of papaya PFL, since an equally divergent but a functional LFY ortholog Needly of Pinus radiata has been reported. Genomic and BAC Southern analyses indicated that there is only one copy of PFL in the papaya genome. In situ hybridization experiments demonstrated that PFL is expressed at a relatively low level in leaf primordia, but it is expressed at a high level in the floral meristem. Quantitative PCR analyses revealed that PFL was expressed in flower buds of all three sex types - male, female, and hermaphrodite with marginal difference between hermaphrodite and unisexual flowers. These data suggest that PFL may play a similar role as LFY in flower development and has limited effect on sex differentiation in papaya. (+info)
'Candidatus Phytoplasma graminis' and 'Candidatus Phytoplasma caricae', two novel phytoplasmas associated with diseases of sugarcane, weeds and papaya in Cuba.
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During 2003, surveys of sugarcane yellow leaf disease and papaya bunchy top-like disease were carried out on plantations in Havana province, Cuba, to determine the roles of weeds and Auchenorrhyncha insects in the epidemiology of these diseases. More than 250 plant and insect samples were collected and indexed by using a nested PCR for phytoplasma 16S rDNA with the generic primer pairs P1/P7 and R16F2n/R16R2. The PCR products were further characterized by restriction fragment length polymorphism using HaeIII, AluI, Sau3AI, Tru9I, HhaI, HpaII and TaqI endonucleases, giving patterns that distinguished them from those of the other reference phytoplasmas analysed. Phylogenetic analysis of 16S rRNA gene sequences identified the phytoplasmas present in sugarcane (Saccharum officinarum L.), Cynodon dactylon L., Conyza canadensis L. Cronq., Sorghum halepense L. Pers., Macroptilium lathyroides L. Urb., Saccharosydne saccharivora (Westwood) and Cedusa spp., and those in papaya (Carica papaya L.) and Empoasca papayae, as two novel provisional phytoplasma species. We propose that these phytoplasmas should be given Candidatus status, as 'Candidatus Phytoplasma graminis' and 'Candidatus Phytoplasma caricae', respectively. (+info)
Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex.
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In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 A and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 A. (+info)
The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein.
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The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive beta-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5-11.0) and temperature (up to 80 degrees C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor. (+info)
Tissue differential expression of lycopene beta-cyclase gene in papaya.
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Carotene pigments in flowers and fruits are distinct features related to fitness advantages such as attracting insects for pollination and birds for seed dispersal. In papaya, the flesh color of the fruit is considered a quality trait that correlates with nutritional value and is linked to shelf-life of the fruit. To elucidate the carotenoid biosynthesis pathway in papaya, we took a candidate gene approach to clone the lycopene beta-cyclase gene, LCY-B. A papaya LCY-B ortholog, cpLCY-B, was successfully identified from both cDNA and bacterial artificial chromosome (BAC) libraries and complete genomic sequence was obtained from the positive BAC including the promoter region. This cpLCY-B shared 80% amino acid identity with citrus LCY-B. However, full genomic sequences from both yellow- and red-fleshed papaya were identical. Quantitative real-time PCR (qPCR) revealed similar levels of expression at six different maturing stages of fruits for both yellow- and red-fleshed genotypes. Further expression analyses of cpLCY-B showed that its expression levels were seven- and three-fold higher in leaves and, respectively, flowers than in fruits, suggesting that cpLCY-B is down-regulated during the fruit ripening process. (+info)
Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents.
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In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device. (+info)