RESUMEN
Easy, quantitative measures of biomolecular heterogeneity and high-stratified phenotyping are needed to identify and characterise complex disease processes at the single-cell level, as well as to predict cell fate. Here, we demonstrate how Raman spectroscopy can be used in the difficult-to-assess case of clonal, bone-derived mesenchymal stromal cells (MSCs) to identify MSC lines and group these according to biological function (e.g., differentiation capacity). Biomolecular stratification is achieved using high-precision measures obtained from representative statistical sampling that also enable quantified heterogeneity assessment. Application to primary MSCs and human dermal fibroblasts shows use of these measures as a label-free assay to classify cell sub-types within complex heterogeneous cell populations, thus demonstrating the potential for therapeutic translation, and broad application to the phenotypic characterisation of other cells.
Asunto(s)
Células Madre Mesenquimatosas/citología , Análisis de la Célula Individual/métodos , Espectrometría Raman/métodos , Diferenciación Celular , Línea Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , FenotipoRESUMEN
A new technique is described for the patterning of cell-guidance cues in synthetic extracellular matrices (ECM) for tissue engineering applications. Using s-tetrazine modified hyaluronic acid (HA), bis-trans-cyclooctene (TCO) crosslinkers and monofunctional TCO conjugates, interfacial bioorthogonal crosslinking was used to covalently functionalize hydrogels as they were synthesized at the liquid-gel interface. Through temporally controlled introduction of TCO conjugates during the crosslinking process, the enzymatic degradability, cell adhesivity, and mechanical properties of the synthetic microenvironment can be tuned with spatial precision. Using human mesenchymal stem cells (hMSCs) and hydrogels with a core-shell structure, we demonstrated the ability of the synthetic ECM with spatially defined guidance cues to modulate cell morphology in a biomimetic fashion. This new method for the spatially resolved introduction of cell-guidance cues for the establishment of functional tissue constructs complements existing methods that require UV-light or specialized equipment.
RESUMEN
Infection by porcine reproductive and respiratory syndrome virus (PRRSV) results in a weak induction of the innate immune response. There are many genes that collectively comprise this response and the extent to which each gene responds to PRRSV infection is unclear and warrants further investigation. To this end, we have utilized real-time PCR using SYBR Green I dye-based detection to quantify transcript abundance of the type I interferons (IFN-alpha and -beta) and IFN-beta transcriptional enhanceasome genes. In MARC-145 cells, both IFN-alpha and -beta transcript abundance were unaffected by PRRSV infection. However, stimulation of MARC-145 cells by exogenous double-stranded RNA, resulted in significant increases in transcript abundance of both IFN-alpha and -beta as well as IFN-beta enhanceasome components, indicating that a type I IFN response could be induced in these cells. The double-stranded RNA induction of type I IFN transcription was significantly inhibited by dual-exposure with PRRSV. These results suggest that PRRSV infection directly interferes with type I IFN transcriptional activation early in its pathway, at the level of IFN-beta gene transcription.
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Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Animales , Línea Celular , Regulación de la Expresión Génica/inmunología , Interferón-alfa/genética , Interferón beta/genética , ARN Mensajero/metabolismo , Porcinos , Factores de Transcripción/metabolismoAsunto(s)
Bovinos/genética , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Genes sry/genética , Haplotipos/genética , Cromosomas Sexuales/genética , Animales , Secuencia de Bases , Cartilla de ADN , Desequilibrio de Ligamiento , Masculino , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Factores de Transcripción , Estados UnidosRESUMEN
Self-medication with analgesics is common and accepted and even recommended by health systems in order to avoid reimbursement. Self-medication, nevertheless, is not an easy task, since making choices is difficult for patients on the basis of the available standard information. Guiding information for patients has to be improved, but also physicians need to be trained how to handle self-medication of their patients. Special attention should be paid to the approval of combination analgesics for the treatment of headache and migraine. There were two major points of discussion during the last decades: possible risks of nephropathy and possible drug-induced overuse. According to a very recent evaluation, analgesic-associated nephropathy appears to have been primarily caused by phenacetin rather than any other single or combination analgesics. Analgesic-induced overuse is also caused by the psychotropic actions of phenacetin in presentations providing rapid absorption, such as powders, rather than by other analgesics or caffeine.
Asunto(s)
Analgésicos no Narcóticos/efectos adversos , Enfermedades Renales/inducido químicamente , Fenacetina/efectos adversos , Automedicación/efectos adversos , Analgésicos no Narcóticos/uso terapéutico , Cafeína/administración & dosificación , Estimulantes del Sistema Nervioso Central/administración & dosificación , Sinergismo Farmacológico , Cefalea/tratamiento farmacológico , Humanos , Educación del Paciente como Asunto , Fenacetina/uso terapéuticoRESUMEN
OBJECTIVE: To determine whether a group of 3 genetic differences in the nonstructural protein (NS1) or 1 genetic difference in the structural protein (VP2) of Aleutian disease parvovirus (ADV) is responsible for an increase in the in vivo replication and pathogenicity of G/U-8, a chimera of ADV-G (nonpathogenic) and ADV-Utah (pathogenic), compared with G/U-10. ANIMALS: 32 eight-month-old female sapphire mink (Mustela vison). PROCEDURE: Chimeric viruses were constructed, propagated in vitro, and used to inoculate mink. Antiviral antibody responses, presence of serum viral nucleic acid, and serum gamma globulin concentrations were monitored for 120 days following inoculation. Histologic examination of the liver, kidneys, spleen, and mesenteric lymph nodes was performed after necropsy. RESULTS: A chimera containing only the 3 amino acid substitutions in NS1 did not elicit measurable responses indicative of replication or pathogenicity in inoculated mink. Serum antiviral antibody responses, frequency of detection of viral nucleic acid in serum, gamma globulin response, and histologic changes in mink inoculated with chimeras containing a valine residue at codon 352 (352V) of VP2 capsid were increased, compared with values from mink inoculated with chimeric viruses that did not contain 352V. CONCLUSIONS AND CLINICAL RELEVANCE: A valine residue at codon 352 in the VP2 capsid protein of ADV affects in vivo viral replication and pathogenicity. This amino acid may be part of an incompletely defined pathogenic determinant of ADV. Further characterization of the pathogenic determinant may allow future development of focused preventive and therapeutic interventions for Aleutian disease of mink.
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Virus de la Enfermedad Aleutiana del Visón/patogenicidad , Enfermedad Aleutiana del Visón/virología , Cápside/genética , Visón/virología , Enfermedad Aleutiana del Visón/sangre , Enfermedad Aleutiana del Visón/patología , Virus de la Enfermedad Aleutiana del Visón/genética , Virus de la Enfermedad Aleutiana del Visón/fisiología , Animales , Anticuerpos Antivirales/sangre , Cápside/química , Proteínas de la Cápside , Quimera/genética , Clonación Molecular , Codón/genética , Contrainmunoelectroforesis/veterinaria , ADN Viral/sangre , Histocitoquímica/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Estadísticas no Paramétricas , gammaglobulinas/análisisRESUMEN
[see reaction]. A protocol for the catalytic asymmetric vinylation of ketone enolates has been developed. Key to the success of this process was the development of new electron-rich chiral monodentate ligands.
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Cetonas/química , Compuestos de Vinilo/química , CatálisisRESUMEN
Chromate-containing primer paints are used to prevent corrosion on metal surfaces. Chromate contains hexavalent chromium (Cr6+), a human carcinogen. The objective of this research was to determine if there is a bias in the fraction of chromate found in various particle sizes generated during primer painting operations. A solvent-based, aviation primer paint was sprayed using a high-volume, low-pressure spray gun. Paint particles were collected and separated by size with seven-stage cascade impactors. It was determined that particles with a mass aerodynamic diameter < 2.0 microm contained significantly less Cr6+ per dry weight of paint than particles > 2.0 microm (P < 0.001). The median concentration of Cr6+ in particles < 2.0 microm is 18 micro g of Cr/mg of dry paint and the median concentration for particles > 2.0 microm is 70 microg of Cr/mg of dry paint. The mixed paint contains 18.75% strontium chromate, which equates to a ratio of 67 microg of Cr/mg of dry paint. Particles > 2.0 microm are more likely to impact in the upper tracheobronchial regions of the lung where mucociliary clearance is relatively rapid. Additionally, chromate emissions from spraying operations may be overestimated because larger particles, which are more easily trapped on an air filter, contain more chromate than the smaller particles, which are more likely to bypass an air filter.
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Carcinógenos Ambientales/análisis , Cromatos/análisis , Cromo/análisis , Pintura , Carcinógenos Ambientales/química , Técnicas de Química Analítica/métodos , Cromatos/química , Cromo/química , Humanos , Exposición por Inhalación , Exposición Profesional , Tamaño de la Partícula , Reproducibilidad de los ResultadosAsunto(s)
Accesibilidad a los Servicios de Salud/legislación & jurisprudencia , Cardiopatías Congénitas/rehabilitación , Adolescente , Adulto , Costos de la Atención en Salud/estadística & datos numéricos , Accesibilidad a los Servicios de Salud/economía , Cardiopatías Congénitas/economía , Humanos , Cobertura del Seguro/economía , Cobertura del Seguro/legislación & jurisprudencia , Programas Controlados de Atención en Salud/economía , Programas Controlados de Atención en Salud/legislación & jurisprudencia , Grupo de Atención al Paciente/economía , Grupo de Atención al Paciente/legislación & jurisprudencia , Rehabilitación Vocacional/economía , Estados UnidosRESUMEN
OBJECTIVE: Debates about the suspected association between kidney disease and use of analgesics have led to concern about whether caffeine could stimulate an undesirable overuse of phenacetin-free combined analgesics. A committee was asked to critically review the pertinent literature and to suggest guides for clinical practice and for consideration of international regulatory authorities. PARTICIPANTS: A group of international scientists, jointly selected by the regulatory authorities of Germany, Switzerland, and Austria and the pharmaceutical industry. EVIDENCE: All invited experts evaluated relevant literature and reports and added further information and comments. CONCLUSIONS: Caffeine has a synergistic effectiveness with analgesics. Although caffeine has a dependence potential, the potential is low. Experimental data regarding dependence potential for caffeine alone may not correspond to the conditions in patients with pain. Withdrawal is not likely to cause stimulation or sustainment of analgesic intake. For drug-induced headache, no single or combined analgesic was consistently identified as causative, and no evidence exists for a special role of caffeine. Strong dependence behavior was observed only in patients using phenacetin-containing preparations, coformulated with antipyretics/analgesics and caffeine. This finding may have led to the impression that caffeine stimulates overuse of analgesics. SUMMARY: Although more experimental and long-term data would be desirable to show possible mechanisms of dependence and to offer unequivocal proof of safety, the committee concluded that the available evidence does not support the claim that analgesics coformulated with caffeine, in the absence of phenacetin, stimulate or sustain overuse.
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Analgésicos/efectos adversos , Cafeína/efectos adversos , Medicina Basada en la Evidencia , Cefalea/inducido químicamente , Fenacetina/efectos adversos , Síndrome de Abstinencia a Sustancias , Sinergismo Farmacológico , Humanos , Trastornos Relacionados con Sustancias/fisiopatologíaRESUMEN
BACKGROUND: The debate on the association between nonphenacetin-containing combined analgesics and renal disease has lasted for several years. METHOD: A peer review committee of scientists, selected jointly by the regulatory authorities of Germany, Switzerland, and Austria and the pharmaceutical industry was asked to critically review data on the relationship between nonphenacetin combined analgesics and nephropathy. RESULTS: The committee regarded epidemiologic evidence on nonphenacetin combined analgesics as inconclusive because of sparse information and substantial methodological problems. The committee also noted that a diagnosis of analgesic-associated nephropathy (AAN) in clinical practice usually depends on information about exposure before or in the early stages of the disease and is seldom accompanied by specific histologic evidence. The morphologic finding of papillary calcification can arise from other conditions and is not specific for AAN. For these reasons, the identification criteria for AAN should be reappraised with scientific methods to validate the diagnostic procedure. In the limited amount of experimental pharmacological data in humans and animals, the committee found no convincing evidence to confirm or refute the hypothesis that nonphenacetin combined analgesics are more nephrotoxic than single formulations. For caffeine taken with combined analgesics, the currently available information is not sufficient to postulate a harmful toxicological effect. CONCLUSION: The committee's two main conclusions were that sufficient evidence is absent to associate nonphenacetin combined analgesics with nephropathy and that new studies should be done to provide appropriate data for resolving the question.
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Analgésicos no Narcóticos/toxicidad , Antiinflamatorios no Esteroideos/toxicidad , Aspirina/toxicidad , Fallo Renal Crónico/inducido químicamente , Acetaminofén/toxicidad , Animales , Cafeína/toxicidad , Combinación de Medicamentos , Humanos , Fenacetina , Inhibidores de Fosfodiesterasa/toxicidadRESUMEN
Functionalized dicyclohexyl- and di-tert-butylphosphinobiphenyl ligands are prepared by the reaction of arylmagnesium halides with benzyne, followed by the addition of a chlorodialkylphosphine. This one-pot procedure is considerably less expensive and time-consuming than the method used previously to prepare such ligands. The cost of introducing the dicyclohexylphosphine group can be decreased by preparing chlorodicyclohexylphosphine from PCl3 and cyclohexylmagnesium chloride, and using the reagent without further purification. The new method is significant, as a variety of ligands can be produced in useful amounts by a procedure that is simple, with starting materials that are relatively inexpensive, and, in most cases, without chromatographic purification.
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Compuestos de Bifenilo/síntesis química , Fosfinas/química , Compuestos de Bifenilo/química , Ligandos , Espectroscopía de Resonancia Magnética , Espectrofotometría InfrarrojaRESUMEN
STUDY OBJECTIVE: The state of Michigan passed Public Act 136 of 1997 requiring Medicaid managed care organizations (MMCOs) to pay for emergency services whenever presenting symptoms constituted an "emergency medical condition." The objective of this study was to evaluate MMCO reimbursement before and after enactment of this state law. METHODS: We conducted a retrospective comparison of reimbursement for lacerations needing repair (identified using Current Procedural Terminology codes from computerized billing data) for 2 time periods (before the state law was applicable [January through March 1998] and after the state law was applicable [April through June 1998]) from MMCO enrollees in 7 different MMCOs presenting to 4 urban emergency departments. Three months after billing submission was allowed for payment. Only refusal of reimbursement was evaluated. Data were analyzed using chi(2) and Fisher's exact test (values of P <.05 were considered significant). RESULTS: The total number of MMCO patients evaluated/total number of ED patients evaluated for the 2 periods was 1,769/32,646 and 3, 376/30,901, respectively (P <.05). The number of MMCO lacerations with no reimbursement/total number of MMCO lacerations for the 2 periods was 4/135 (3%) and 78/196 (40%), respectively (P <.001). CONCLUSION: Reimbursement by MMCOs for a procedure chosen to reflect a state-defined "emergency medical condition" is inadequate and significantly decreased during the 2 periods, with a significant increase in MMCO patients evaluated.
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Servicio de Urgencia en Hospital/economía , Servicio de Urgencia en Hospital/legislación & jurisprudencia , Reembolso de Seguro de Salud/economía , Reembolso de Seguro de Salud/legislación & jurisprudencia , Programas Controlados de Atención en Salud/economía , Programas Controlados de Atención en Salud/legislación & jurisprudencia , Medicaid/economía , Medicaid/legislación & jurisprudencia , Distribución de Chi-Cuadrado , Humanos , Michigan , Mecanismo de Reembolso , Estudios Retrospectivos , Estados Unidos , Heridas y Lesiones/terapiaRESUMEN
STUDY OBJECTIVE: To evaluate the reimbursement difference for Medicaid managed care organization (MMCO) enrollees compared with Medicaid enrollees for emergency department patients with disease conditions that appear to meet the "prudent layperson" definition of an emergency medical condition. METHODS: This study used a retrospective reimbursement review of computerized billing data of reimbursement denials for 4 procedures (using Current Procedural Terminology codes for endotracheal intubation, cardiopulmonary resuscitation, central line placement, and lumbar puncture) and 1 International Classification of Diseases, ninth revision condition (chest pain) on MMCO patients from 7 MMCOs compared with standard Medicaid patients presenting to 4 EDs during a 6-month period (January through June 1998). Exclusion criteria were late bills that did not allow at least 90 days for payment and bills submitted on behalf of patients that were not covered at the time of service by Medicaid or MMCO. Data were analyzed using Fisher's exact test. RESULTS: The total number of MMCO and Medicaid patients evaluated/total ED patients evaluated was 5,153/63,552 and 6,020/63, 552, respectively. The number of nonreimbursed procedures/total number of procedures performed on MMCO and Medicaid patients was 35/93 and 14/88, respectively (P <.05). The number of nonreimbursed chest pain patients/total chest pain patients evaluated for MMCO and Medicaid enrollees was 65/277 and 12/199, respectively (P <.05). CONCLUSION: MMCOs reimburse significantly less than Medicaid does for ED patients with conditions that a prudent layperson would consider an emergency.
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Servicio de Urgencia en Hospital/economía , Reembolso de Seguro de Salud/economía , Programas Controlados de Atención en Salud/economía , Medicaid/economía , Intervalos de Confianza , Servicio de Urgencia en Hospital/legislación & jurisprudencia , Humanos , Reembolso de Seguro de Salud/legislación & jurisprudencia , Programas Controlados de Atención en Salud/legislación & jurisprudencia , Medicaid/legislación & jurisprudencia , Michigan , Oportunidad Relativa , Mecanismo de Reembolso , Estudios Retrospectivos , Estados UnidosAsunto(s)
Enfermedad de Alzheimer/economía , Costo de Enfermedad , Servicios de Salud para Ancianos/economía , Anciano , Enfermedad de Alzheimer/clasificación , Enfermedad de Alzheimer/epidemiología , Financiación Gubernamental/economía , Financiación Personal/economía , Humanos , Institucionalización/economía , SueciaRESUMEN
To further investigate neural effects on leptin and uncoupling proteins (UCPs), we studied in vivo perturbations intended to block adrenergic input to peripheral tissues. We examined plasma leptin, leptin mRNA, and adipose and muscle UCP subtype mRNA in rats treated with alpha-methyl-p-tyrosine methyl ester (AMPT-ME), which inhibits catecholamine synthesis and 6-hydroxydopamine (6HDA), which is toxic to catecholinergic nerve terminals but, unlike AMPT-ME, does not enter the central nervous system. Intraperitoneal AMPT-ME, 250 mg/kg, was administered at 1800 and 0700 the following day, and rats were killed at 1200-1400. All rats were fasted with free access to water during this time. Intraperitoneal AMPT-ME increased plasma leptin by 15-fold, increased interscapular brown adipose tissue (IBAT) and epididymal fat leptin mRNA by 2- to 2.5-fold, and also increased plasma insulin and glucose concentrations. Intraperitoneal AMPT-ME decreased IBAT UCP-3 mRNA to 40% of control, while it increased epididymal adipose UCP-3 mRNA approximately twofold. Intravenous AMPT-ME, 250 mg/kg, administered to conscious rats for 5 h decreased lumbar sympathetic nerve activity, increased plasma leptin (5.89 +/- 1.43 compared with 2.75 +/- 0.31 ng/ml in vehicle-treated rats, n = 7, P < 0.05), and decreased cardiac rate with no sustained change in blood pressure. Intraperitoneal 6HDA, 100 mg/kg, as a single dose at 1800, increased plasma leptin approximately twofold after 18-20 h, increased IBAT (but not epididymal fat) leptin mRNA by two- to threefold, and decreased IBAT UCP-3 mRNA to 30-40% of control. Neither AMPT-ME nor 6HDA significantly altered mRNA encoding gastrocnemius muscle UCP-3, IBAT UCP-1, or IBAT and epididymal UCP-2. In summary, AMPT-ME and 6HDA increased plasma leptin and upregulated leptin mRNA expression. AMPT-ME also resulted in complex tissue and subtype-specific modulation of adipose UCP mRNA. These data are consistent with interaction between leptin and sympathetic nerve activity (SNA) in regulation of fat cell energy utilization. However, the in vivo modulation of leptin and UCPs appears complex and, beyond a causal effect of SNA per se, may depend on concurrent changes in plasma insulin, glucose, and circulatory hemodynamics.
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Proteínas Portadoras/metabolismo , Ayuno/fisiología , Leptina/metabolismo , Proteínas de la Membrana/metabolismo , Inhibición Neural/fisiología , Sistema Nervioso Simpático/fisiología , Tejido Adiposo/metabolismo , Adrenérgicos/farmacología , Animales , Canales Iónicos , Leptina/sangre , Leptina/genética , Metiltirosinas/farmacología , Proteínas Mitocondriales , Oxidopamina/farmacología , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Proteína Desacopladora 1RESUMEN
Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.
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Virus de la Enfermedad Aleutiana del Visón/fisiología , Enfermedad Aleutiana del Visón/virología , Cápside/fisiología , Replicación Viral , Enfermedad Aleutiana del Visón/patología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de la Cápside , Gatos , Hígado/patología , Hígado/virología , Visón , Datos de Secuencia Molecular , Análisis de SecuenciaRESUMEN
Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (=30 degrees C) and virulence plasmid-encoded Yop secretion (37 degrees C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the Y. enterocolitica virulence plasmid, pYV, undergoes a conformational transition between 30 and 37 degrees C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including virF, the positive activator of virulence genes. These bends are detectable at 30 degrees C but melt at 37 degrees C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from Y. enterocolitica were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the Y. enterocolitica pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in Y. enterocolitica and other enteric pathogens.
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ADN Bacteriano/química , Conformación de Ácido Nucleico , Plásmidos/genética , Virulencia/genética , Yersinia enterocolitica/genética , Cloroquina , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Electroforesis en Gel de Agar , Electroforesis en Gel Bidimensional , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Temperatura , Yersinia enterocolitica/patogenicidadRESUMEN
The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.