RESUMEN
The increase in the consumption of fresh produce has correlated with a rise in the number of reported foodborne illnesses. To identify potential risk factors associated with postharvest practices, the present study employed multilocus sequence typing (MLST) for the genotypic classification of Escherichia coli isolates recovered from three sources sampled at seven operational stages in a cantaloupe packinghouse in Northwestern Mexico. The MLST analysis results indicated that the E. coli isolates were classified into 18 different sequence types (ST), and 11 of these STs were found to be novel. ST-171 was the predominant type and was found in 19% (7/36) of the recovered isolates. Interestingly, the novel ST-827 was found to be significantly associated with isolates recovered from workers' hands, sampled during final postwash stages. Further phylogenetic analyses to examine the relatedness of the STs revealed genetic heterogeneity. Fourteen of the identified STs were assigned to known clonal groups, while the remaining four novel STs were distinct and did not cluster with any clonal group. The present study has provided the first evidence indicating that several sources from distinct operational stages in a cantaloupe packinghouse may contribute to a genotypic and phylogenetic diverse set of E. coli isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Packinghouses can be considered as a potential source of microbial contamination. Using multilocus sequence typing, this study identified a genotypic and phylogenetic diverse set of Escherichia coli isolates recovered from the surfaces of cantaloupes, workers' hands and processing equipment at a cantaloupe packinghouse. A total of 61% of the sequence types identified were novel, and a distinct sequence type, ST-827, was significantly associated with worker's hands, sampled during the final postwash operational stages in the packinghouse. These findings serve as a baseline to identify potential sources of microbial contamination at distinct operational stages in a cantaloupe packinghouse.
Asunto(s)
Cucumis melo/microbiología , Escherichia coli/genética , Contaminación de Alimentos , Embalaje de Alimentos , Variación Genética , Técnicas de Tipificación Bacteriana , Escherichia coli/aislamiento & purificación , Genotipo , Humanos , México , Tipificación de Secuencias Multilocus , FilogeniaRESUMEN
Although there is evidence that the thalamus plays a remarkable role in pain processing few in vivo studies on the thalamic neurochemical correlates of pain have been done. In the present experiments a combination of capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) and microdialysis in freely moving rats was used to measure extracellular arginine, glutamate and aspartate in the thalamus during the formalin test. Microdialysis probes were implanted in the left ventral posterolateral (VPL) nucleus of the thalamus in rats. Samples were collected every 30 s, derivatized with fluorescein isothyocyanate and injected into a CZE-LIF instrument. After nine baseline samples, a subcutaneous formalin (5%, 50 microl) injection in the right hind paw caused an increase of arginine, glutamate and aspartate that lasted for about 3 min. These increases were calcium and nerve impulse dependent. These results indicate that the release of arginine, glutamate and aspartate may mediate rapid pain neural transmission in the VPL nucleus of the thalamus.
Asunto(s)
Aminoácidos/metabolismo , Nociceptores/fisiología , Núcleos Talámicos Ventrales/metabolismo , Animales , Arginina/metabolismo , Ácido Aspártico/metabolismo , Electroforesis Capilar , Espacio Extracelular/metabolismo , Ácido Glutámico/metabolismo , Masculino , Microdiálisis , Dolor/metabolismo , Dimensión del Dolor , Ratas , Ratas Wistar , Transmisión Sináptica/fisiologíaRESUMEN
A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 microM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 microM. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.
Asunto(s)
Proteínas Bacterianas , Técnicas Biosensibles , Genes Reporteros , Sacarosa/metabolismo , Erwinia/genética , Erwinia/metabolismo , Fabaceae/microbiología , Regulación Bacteriana de la Expresión Génica , Proteínas Fluorescentes Verdes , Operón Lac/genética , Operón Lac/fisiología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Plantas Medicinales , Plásmidos/genética , Porinas/genética , Porinas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transcripción GenéticaRESUMEN
We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC.
Asunto(s)
Carboxiliasas/genética , Erwinia/enzimología , Fabaceae/metabolismo , Genes Bacterianos , Ácidos Indolacéticos/metabolismo , Transcripción Genética , Erwinia/genética , Hojas de la Planta , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2'), 2C (2") and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.
Asunto(s)
Empalme Alternativo , Antígenos de Superficie/genética , Variación Genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/química , Encéfalo/metabolismo , Células COS , Membrana Celular/metabolismo , Clonación Molecular , Regulación de la Expresión Génica , Células HeLa , Humanos , Riñón/metabolismo , Hígado/metabolismo , Masculino , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares/metabolismo , Sintaxina 1 , Testículo/metabolismo , Transcripción Genética , TransfecciónRESUMEN
The pH and trafficking of recycling endosomes have previously been studied using transferrin. We have used another approach, one in which the vesicle transport protein cellubrevin was appended with a luminal IgG epitope to allow targeting of fluorescein-5'-isothiocyanate (FITC)-labeled anti-IgG F(ab) antibodies to the recycling endosomes in living cells. FITC-F(ab) was specifically internalized by COS cells transfected with cellubrevin-Ig, which at steady state accumulated in a pericentriolar region similar to rhodamine-transferrin. Confocal microscopic analysis showed that endosome labeling by these two markers was heterogeneous. This differential distribution was not induced by the IgG tag, since endogenous Cb and Tf were also partitioned into separate endosomal populations. We used fluorescence ratio imaging of internalized FITC-F(ab) to measure the pH of cellubrevin-enriched recycling endosomes (pHCb) and FITC-transferrin to measure the pH of transferrin-enriched recycling endosomes (pHTf). In COS cells, cellubrevin endosomes (mean pHCb 6.1 +/- 0.05; range, 5.2-6.6) were more acidic than transferrin endosomes (mean pHTf 6.5 +/- 0.05; range, 5.6-7.2). Similar results were obtained in Chinese hamster ovary cells. Treatment with the vacuolar H+-ATPase inhibitor bafilomycin A1 caused pHTf to increase (DeltapHTf = 1.2 pH units) to a greater extent than pHCb (DeltapHCb = 0.5 pH units). Furthermore, inhibition of the Na+/K+-ATPase by ouabain or acetylstrophanthidin caused pHTf to decrease by 0.6 pH units but had no effect on pHCb. Based on the combination of these morphological and functional data, we suggest that the recycling endosomes are heterogeneous in their biochemical compositions, ion transport properties, and pH values.
Asunto(s)
Endosomas/fisiología , Macrólidos , Proteínas de la Membrana/metabolismo , Animales , Antibacterianos/farmacología , Células COS/citología , Epítopos/inmunología , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescencia , Técnica del Anticuerpo Fluorescente , Concentración de Iones de Hidrógeno , Inmunoglobulina G/inmunología , Microscopía Confocal , Ouabaína/farmacología , Estrofantidina/farmacología , Transfección/genética , Transferrina/metabolismo , Proteína 3 de Membrana Asociada a VesículasRESUMEN
Field recordings of evoked excitatory postsynaptic potentials (pEPSPs) were carried out in the granule cell stratum moleculare following stimulation of the perforant path in rat hippocampal slices. Under control conditions tetanic stimulation produced long-term potentiation (LTP) as measured by an increase in the initial slope of the pEPSPs that lasted for at least 1 h. LTP experiments were repeated with 0.5, 5.0, 50, or 500 nM angiotensin II (AII) present in the bath at the time of tetanization. Induction of LTP was blocked by 50 nM AII; however, normal baseline responses were not affected. At the highest dose tested, 500 nM, a decrease in the amplitude and slope of baseline pEPSPs was observed. When the AII AT1 receptor antagonist losartan was present in the bath AII inhibition of LTP was blocked. The application of losartan alone had no effect on LTP expression. These findings support previous results from in vivo studies demonstrating that activation of AT1 receptors in the dentate gyrus blocks the induction of LTP at the perforant path-granule cell synapse.