RESUMEN
A selective DNA pooling approach was applied to identify QTL for conjugated linoleic acid (CLA), vaccenic acid (VA) and Δ(9) -desaturase (D9D) milk content in Italian Brown Swiss dairy cattle. Milk samples from 60 animals with higher values (after correction for environmental factors) and 60 animals with lower values for each of these traits from each of five half-sib families were pooled separately. The pools were genotyped using the Illumina BovineSNP50 BeadChip. Sire allele frequencies were compared between high and low tails at the sire and marker level for SNPs for which the sires were heterozygous. An r procedure was implemented to perform data analysis in a selective DNA pooling design. A correction for multiple tests was applied using the proportion of false positives among all test results. BTA 19 showed the largest number of markers in association with CLA. Associations between SNPs and the VA and Δ(9) -desaturase traits were found on several chromosomes. A bioinformatics survey identified genes with an important role in pathways for milk fat and fatty acids metabolism within 1 Mb of SNP markers associated with fatty acids contents.
Asunto(s)
Bovinos/genética , Ácidos Linoleicos Conjugados/genética , Ácidos Oléicos/genética , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Estearoil-CoA Desaturasa/genética , Animales , Bovinos/metabolismo , Femenino , Frecuencia de los Genes , Ácidos Linoleicos Conjugados/metabolismo , Glándulas Mamarias Animales/enzimología , Glándulas Mamarias Animales/metabolismo , Leche/química , Ácidos Oléicos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinaria , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm(2), 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (>80%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.
Asunto(s)
Células/metabolismo , ADN/administración & dosificación , Terapia Genética/métodos , Transfección/métodos , Terapia por Ultrasonido , Albúminas/uso terapéutico , Animales , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Medios de Contraste , ADN/análisis , Fluorocarburos/uso terapéutico , Proteínas Fluorescentes Verdes/genética , Microscopía de Fuerza Atómica , Microscopía Confocal , MicroesferasRESUMEN
The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.
Asunto(s)
Cromatina/metabolismo , Citoprotección , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/metabolismo , Colesterol/metabolismo , Cromatina/genética , Cromatina/ultraestructura , Cristalización , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Escherichia coli/citología , Escherichia coli/ultraestructura , Iones , Magnesio/farmacología , Microscopía Electrónica , Dispersión de Radiación , Rayos XRESUMEN
The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli. We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.
Asunto(s)
Reparación del ADN , Escherichia coli/genética , Rec A Recombinasas/química , Respuesta SOS en Genética , Daño del ADNRESUMEN
Natural aroma compounds are of major interest to the flavor and fragrance industry. Due to the limited sources for natural aromas, there is a growing interest in developing alternative sources for natural aroma compounds, and in particular aromatic aldehydes. In several microbial species aromatic aldehydes are detected as intermediates in the degradation pathway of phenylpropanoids. Thus, bioconversion of phenylpropanoids is one possible route for the production of these aroma compounds. The present work describes the isolation of microbial strains, capable of producing vanillin from isoeugenol. Bacterial strains isolated from soil, were screened for their ability to transform isoeugenol to vanillin. One of these strains, strain B2, was found to produce high amounts of vanillin when grown in the presence of isoeugenol, and was also capable of growing on isoeugenol as the sole carbon source. Based on its fatty acids profile, strain B2 was identified as a Bacillus subtilis sp. The bioconversion capabilities of strain B2 were tested in growing cultures and cell free extracts. In the presence of isoeugenol, a growing cultures of B. subtilis B2 produced 0.61 g l-1 vanillin (molar yield of 12.4%), whereas cell free extracts resulted in 0.9 g l-1 vanillin (molar yield of 14%).
Asunto(s)
Bacillus subtilis/aislamiento & purificación , Bacillus subtilis/metabolismo , Benzaldehídos/metabolismo , Eugenol/análogos & derivados , Aromatizantes/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Biotecnología/métodos , Sistema Libre de Células , Eugenol/metabolismo , Ácidos Grasos/análisis , Microbiología de Alimentos , Espectrometría de Masas , Mutación , PerfumesRESUMEN
We investigated the development of secretory granules in the pancreatic acinar cells of normal (C57BL/6J +/+) and beige (C57BL/6J Lystbg/Lystbg) mice by analyzing the distribution of 3H label in pancreatic acinar cells after a pulse of [3H]glycine administered in vivo. The results provide quantitative confirmation of the hypothesis that the maturation of condensing vacuoles/immature granules to mature granules in pancreatic acinar cells is associated with a significant volume reduction. Beige mice differ from control mice by exhibiting a more rapid distribution of 3H label from the rough endoplasmic reticulum-rich cytoplasm to the secretory granules and a slightly faster rate of maturation of 3H-labeled granules. Beige mouse pancreatic acinar cells also exhibited, as early as 1 h after pulsing with [3H]glycine, a much higher proportion of 3H-labeled secretory granules than did the cells of control mice. These findings identify additional abnormalities in secretory granule formation in pancreatic acinar cells which are related to the beige (Lystbg) mutation and provide support for the hypothesis that beige mice exhibit an abnormal pattern of granule-granule fusion.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , Glicina/metabolismo , Páncreas/fisiología , Animales , Autorradiografía , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Retículo Endoplásmico Rugoso/metabolismo , Cinética , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Microscopía Electrónica , Páncreas/metabolismo , Páncreas/ultraestructura , Especificidad de la EspecieRESUMEN
High-pressure freezing (HPF) is currently the only method which enables adequate cryoimmobilization of biological samples thick enough to describe the bulk of the sample. In the current state of HPF instrumentation and preparation methods, the technique has not yet reached its full potential. While suspensions can be prepared easily for HPF, tissue preparation is restricted by the need to compromise between different requirements and difficulties. (i) In order to achieve optimal freezing quality, very thin samples are required. (ii) There is mechanical difficulty in cutting such thin samples without distorting the organization of the tissue. (iii) The cutting and the succeeding preparation steps of small samples require long handling times (minutes), which may result in physiological and hence structural alterations. Computerized heat transfer simulations are presented which confirm that the efficiency of heat extraction from cylindrical samples contained within thin-walled metal tubes is higher than from standard flat discoid samples sandwiched between relatively thick aluminium platelets. Based on this fact, we developed a prototype of a new microbiopsy device which enables the quick excision of such cylinders of soft tissues. The device utilizes sharp gold needles of an inner diameter of 200 microm and wall thickness of 50 microm. The frozen sample contained in the soft gold needle permits all the manipulations needed for conventional cryo-preparation techniques for electron microscopy (e.g. cryo-sectioning, freeze-fracturing, freeze-substitution).
Asunto(s)
Criopreservación/instrumentación , Técnica de Fractura por Congelación , Secciones por Congelación , Microscopía Electrónica/métodos , Manejo de Especímenes/instrumentación , Animales , Simulación por Computador , Criopreservación/métodos , Diseño de Equipo , Congelación , Riñón/ultraestructura , Miocardio/ultraestructura , Presión , Ratas , Manejo de Especímenes/métodos , Conductividad TérmicaRESUMEN
Time resolved fluorimetry was employed to monitor the geminate recombination between proton and excited pyranine anion locked, together with less than 30 water molecules, inside the heme binding site of Apomyoglobin (sperm whale). The results were analyzed by a numerical reconstruction of the differential rate equation for time-dependent diffusion controlled reaction with radiating boundaries using N. Agmon's procedure (Huppert, Pines, and Agmon, 1990, J. Opt. Soc. Am. B., 7:1541-1550). The analysis of the curve provided the effective dielectric constant of the proton permeable space in the cavity and the diffusion coefficient of the proton. The electrostatic potential within the cavity was investigated by the equations given by Gilson et al. (1985, J. Mol. Biol., 183:503-516). According to this analysis the dielectric constant of the protein surrounding the site is epsilon prot < or = 6.5. The diffusion coefficient of the proton in the heme binding site of Apomyoglobin-pyranine complex is D = 4 x 10(-5) cm2/s. This value is approximately 50% of the diffusion coefficient of proton in water. The lower value indicates enhanced ordering of water in the cavity, a finding which is corroborated by a large negative enthropy of binding delta S0 = -46.6 cal.mole-1 deg-1. The capacity of a small cavity in a protein to retain a proton had been investigated through the mathematical reconstruction of the dynamics. It has been demonstrated that Coulombic attraction, as large as delta psi of energy coupling membrane, is insufficient to delay a free proton for a time frame comparable to the turnover time of protogenic sites.
Asunto(s)
Apoproteínas/química , Mioglobina/química , Animales , Arilsulfonatos , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Difusión , Electroquímica , Colorantes Fluorescentes , Hemo/química , Protones , Espectrometría de Fluorescencia , Termodinámica , Agua/químicaRESUMEN
The reaction mechanism and the dynamic aspects of protonation of a defined moiety located inside an aqueous cavity in a protein were monitored by time resolved spectroscopy using the pyranine apomyoglobin complex as a model (Shimoni, Tsfadia, Nachliel, and Gutman, 1993, Biophys. J. 64:472-479). The reaction was synchronized by a short laser pulse and the reprotonation of the ground state pyranine anion (phi O-) was monitored, in the microsecond time scale, by its transient absorption at 457 nm. The observed signal was reconstructed by a numeric solution of nonlinear, coupled differential equations which account for the direct reaction of phi O- with bulk proton and by proton transfer from the nearby amino acids: His 64, Asp 44, Asp 60, and Glu 59. A unique combination of rate constant was obtained which quantitates the contribution of each pathway to the overall relaxation process. In the first phase of the dynamics phi O- abstracts a proton from the nearby protonated histidine. The bulk proton interacts preferentially with the cluster of three carboxylates and immediately shuttled to the deprotonated histidine. The high proximity of the reactive groups and the strong electrostatic forces operating inside the heme binding cavity render the rate of proton transfer in the site ultrafast.