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OBJECTIVE: To evaluate the longitudinal reproducibility and variations of cartilage T1ρ and T2 measurements using different coils, MR systems and sites. METHODS: Single-Site study: Phantom data were collected monthly for up to 29 months on four GE 3T MR systems. Data from phantoms and human subjects were collected on two MR systems using the same model of coil; and were collected on one MR system using two models of coils. Multi-site study: Three participating sites used the same model of MR systems and coils, and identical imaging protocols. Phantom data were collected monthly. Human subjects were scanned and rescanned on the same day at each site. Two traveling human subjects were scanned at all three sites. RESULTS: Single-Site Study: The phantom longitudinal RMS-CVs ranged from 1.8% to 2.7% for T1ρ and 1.8-2.8% for T2. Significant differences were found in T1ρ and T2 values using different MR systems and coils. Multi-Site Study: The phantom longitudinal RMS-CVs ranged from 1.3% to 2.6% for T1ρ and 1.2-2.7% for T2. Across three sites (n = 16), the in vivo scan-rescan RMS-CV was 3.1% and 4.0% for T1ρ and T2, respectively. Phantom T1ρ and T2 values were significantly different between three sites but highly correlated (R > 0.99). No significant difference was found in T1ρ and T2 values of traveling controls, with cross-site RMS-CV as 4.9% and 4.4% for T1ρ and T2, respectively. CONCLUSION: With careful quality control and cross-calibration, quantitative MRI can be readily applied in multi-site studies and clinical trials for evaluating cartilage degeneration.
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Cartílago Articular/patología , Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/diagnóstico , Fantasmas de Imagen , Voluntarios Sanos , Humanos , Procesamiento de Imagen Asistido por Computador , Estudios Longitudinales , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/métodos , Reproducibilidad de los ResultadosRESUMEN
SUMMARY: Subchondral trabecular bone structure was analyzed in knee osteoarthritis (OA) patients using 3-T MRI to investigate structural features of subchondral trabecular bone of knee OA. With OA progression, osteoporotic changes were observed in the lateral joint, showing a higher correlation than sclerotic changes in the medial joint. INTRODUCTION: To investigate structural features of subchondral trabecular bone of knee osteoarthritis (OA). METHODS: Sixty knees with KL grade 0-4 (all female) were examined. Fast imaging employing steady-state acquisition-cycled phases (FIESTA-c) and FatSat Spoiled gradient recalled acquisition in the steady state (SPGR) images were acquired by 3-T MRI. At four sites (the medial femur, medial tibia, lateral femur, and lateral tibia), subchondral trabecular bone structure was analyzed by FIESTA-c imaging, cartilage area was measured by SPGR imaging, and their correlation was analyzed. In addition, the subjects were classified into four groups from the cartilage area measured by SPGR imaging, and subchondral trabecular bone structure in each group was compared. RESULTS: As cartilage area decreased in the medial joint, bone volume fraction and trabecular thickness in the medial tibia increased, and bone volume fraction, trabecular thickness, number, and connectivity in the lateral femur and lateral tibia decreased (r ≥ 0.4 or ≤-0.4, p ≤ 0.001). Compared to medially, the changes laterally showed a higher correlation. When the medial-lateral ratio of trabecular thickness in the tibia was determined, it had the highest correlation coefficient (r=-0.7, p < 0.001). These changes were not significantly detected in the early stage. CONCLUSIONS: To more sensitively detect OA changes in subchondral trabecular bone structure, a focus on osteoporotic changes in the lateral joint and the medial-lateral ratio would be useful. Detectability of early OA remains unknown, but based on a strong correlation with the degree of OA progression, trabecular structural analysis of subchondral bone may be a useful parameter to evaluate OA severity and evaluate treatment.
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Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/complicaciones , Osteoporosis/etiología , Anciano , Anciano de 80 o más Años , Cartílago Articular/patología , Estudios Transversales , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Persona de Mediana Edad , Osteoartritis de la Rodilla/diagnóstico , Osteoporosis/diagnóstico , Tibia/patologíaRESUMEN
Some patients with cirrhosis experience rupture of venous varices before operation, and liver transplantation is a therapy of last resort for these patients. However, we have experienced two cases of intraoperative rupture in whom no abnormalities of the venous varices were seen on endoscopy before operation. One patient with ruptured gastrointestinal varices was treated by direct surgical ligation and the other with ruptured oesophageal gastric varices, spontaneously recovered with a Sengstaken-Blakemore tube. These cases suggest that acute variceal haemorrhage should always be considered as a possibility during living-donor liver transplantation in patients with a history of upper gastrointestinal bleeding. Careful observation of the nasogastic tube is important during clamping of the hepatic portal vein.
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Várices Esofágicas y Gástricas/complicaciones , Hemorragia Gastrointestinal/etiología , Trasplante de Hígado/efectos adversos , Donadores Vivos , Constricción , Humanos , Masculino , Persona de Mediana Edad , Vena PortaRESUMEN
BACKGROUND: Thymus-and-activation-regulated chemokine (TARC; CCL17) is related to both allergy and pregnancy, but the relationships of maternal and umbilical cord blood CCL17 to atopic dermatitis (AD) development have not yet been examined. Objective Seventy paired full-term and normal vaginal delivery newborns and their mothers were enrolled in this study. METHODS: To elucidate the pathogenesis and fetomaternal inheritance of AD in infancy, CCL17, IFN-γ-inducible protein 10 kDa (IP-10; CXCL10), soluble HLA-G (sHLA-G), IgE and eosinophil counts were examined using sera from 70 paired umbilical cord and maternal blood samples. RESULTS: Serum CCL17 (r(s) =0.340, P<0.001) and sHLA-G (r(s) =0.600, P<0.001) levels showed high correlations between umbilical cord and maternal blood. Umbilical cord serum levels of CCL17 from neonates destined to develop AD in infancy were higher than in those from neonates who showed no signs of AD during infancy (median 1586.9 vs. 819.6 pg/mL, P<0.001). Serum levels of CCL17 were higher in mothers with AD than in those without AD (median 909.6 vs. 214.1 pg/mL, P<0.001). High umbilical cord serum levels of CCL17 were associated with infantile AD development even in 62 neonates born to mothers without AD (median 1514.4 vs. 740.6 pg/mL, P<0.001) and 38 neonates born to mothers with no allergies (median 1624.2 vs. 740.6 pg/mL, P<0.001). The summary estimates for umbilical cord serum CCL17 in the diagnosis of infantile AD were: sensitivity 85.7% (95% confidence interval: 72.8-98.7), specificity 73.8% (60.5-87.1), positive predictive value 68.6% (53.2-84.0) and negative predictive value 88.6% (78.0-99.1). CONCLUSION AND CLINICAL RELEVANCE: These findings suggest that the umbilical cord blood CCL17 may be involved in the pathogenesis of infantile AD and in fetomaternal inheritance. Serum levels of CCL17 from umbilical cord blood may be a predictive marker for AD in infancy.
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Quimiocina CCL17/sangre , Dermatitis Atópica/sangre , Dermatitis Atópica/congénito , Sangre Fetal/química , Adulto , Edad de Inicio , Biomarcadores/sangre , Estudios de Casos y Controles , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Valor Predictivo de las Pruebas , Pronóstico , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Re-expansion pulmonary oedema is a rare complication caused by rapid re-expansion of a chronically collapsed lung. Several cases of pulmonary oedema associated with one-lung ventilation (OLV) have been reported recently. Elevated levels of pro-inflammatory cytokines in pulmonary oedema fluid are suggested to play important roles in its development. Activation of cytokines after re-expansion of collapsed lung during OLV has not been thoroughly investigated. Here we investigated the effects of re-expansion of the collapsed lung on pulmonary oedema formation and pro-inflammatory cytokine expression. METHODS: Lungs isolated from female white Japanese rabbits were perfused and divided into a basal (BAS) group (n=7, baseline measurement alone), a control (CONT) group (n=9, ventilated without lung collapse for 120 min) and an atelectasis (ATEL) group (n=9, lung collapsed for 55 min followed by re-expansion and ventilation for 65 min). Pulmonary vascular resistance (PVR) and the coefficient of filtration (Kfc) were measured at baseline and 60 and 120 min. At the end of perfusion, bronchoalveolar lavage fluid/plasma protein ratio (B/P), wet/dry lung weight ratio (W/D) and mRNA expressions of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and myeloperoxidase (MPO) were determined. RESULTS: TNF-alpha and IL-1beta mRNA were significantly up-regulated in lungs of the ATEL group compared with BAS and CONT, though no significant differences were noted in PVR, Kfc, B/P and W/D within and between groups. MPO increased at 120 min in CONT and ATEL groups. CONCLUSION: Pro-inflammatory cytokines were up-regulated upon re-expansion and ventilation after short-period lung collapse, though no changes were noted in pulmonary capillary permeability.
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Permeabilidad Capilar/fisiología , Citocinas/análisis , Atelectasia Pulmonar/terapia , Edema Pulmonar/etiología , Animales , Presión Sanguínea/fisiología , Proteínas Sanguíneas/análisis , Northern Blotting/métodos , Líquido del Lavado Bronquioalveolar , Femenino , Expresión Génica/fisiología , Interleucina-1/análisis , Pulmón/fisiopatología , Tamaño de los Órganos , Peroxidasa/análisis , Atelectasia Pulmonar/fisiopatología , Edema Pulmonar/fisiopatología , ARN Mensajero/análisis , Conejos , Factor de Necrosis Tumoral alfa/análisis , Regulación hacia Arriba , Resistencia Vascular/fisiologíaRESUMEN
Some patients with Mycoplasma pneumoniae infection are clinically resistant to antibiotics such as erythromycin, clarithromycin, or clindamycin. We isolated M. pneumoniae from such patients and found that one of three isolates showed a point mutation in the 23S rRNA gene. Furthermore, 141 EM-sensitive clinical isolates of M. pneumoniae were cultured in broth medium containing 100 microg/ml of erythromycin (EM). Among 11 EM-resistant strains that grew in the medium, point mutations in the 23S rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM-resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.
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Antibacterianos/farmacología , Eritromicina/farmacología , Mycoplasma pneumoniae/efectos de los fármacos , Neumonía por Mycoplasma/microbiología , Secuencia de Bases , Farmacorresistencia Bacteriana/genética , Humanos , Datos de Secuencia Molecular , Mutación , Mycoplasma pneumoniae/genética , ARN Bacteriano/genética , ARN Ribosómico 23S/genéticaRESUMEN
BACKGROUND: Although potassium channels are thought to be responsible for the initiation of hypoxic pulmonary vasoconstriction (HPV), their role in the HPV-inhibitory effect of volatile anesthetics is unclear. The current study tested if the HPV-inhibitory effect of isoflurane and sevoflurane can be affected by changing the potassium-channel opening status with specific potassium-channel inhibitors in isolated rabbit lungs. METHODS: Isolated rabbit lungs were divided into eight groups (n = 6 each in isoflurane groups and n = 8 in sevoflurane groups): those receiving no inhibitor treatment = control-isoflurane and control-sevoflurane groups; those treated with an adenosine triphosphate-sensitive potassium (K(ATP))-channel inhibitor, glibenclamide = glibenclamide-isoflurane and glibenclamide-sevoflurane groups; those treated with a high-conductance calcium-activated potassium (K(Ca))-channel inhibitor, iberiotoxin = iberiotoxin-isoflurane and iberiotoxin-sevoflurane groups; and those treated with a voltage-sensitive potassium (Kv)-channel inhibitor, 4-aminopyridine = 4-aminopyridine-isoflurane and 4-aminopyridine-sevoflurane groups. The effect of anesthetic on HPV was tested by exposure of the lungs to isoflurane at a concentration of 0, 0.5, 1, or 2 minimum alveolar concentration, or to sevoflurane at a concentration of 0, 0.5, 1, or 1.62 minimum alveolar concentration. The relation between anesthetic concentrations and the HPV response was analyzed by the Wagner equation. RESULTS: The inhibition of Kv channels by 4-aminopyridine and K(Ca) channels by iberiotoxin augmented the HPV response. The isoflurane-induced attenuation of HPV was attenuated by voltage-sensitive potassium-channel inhibition with 4-aminopyridine, potentiated by K(Ca)-channel inhibition with iberiotoxin, but not affected by K(ATP)-channel inhibition with glibenclamide. The sevoflurane-induced attenuation of HPV was not affected by any of the potassium-channel inhibitors. CONCLUSIONS: Isoflurane may modulate the HPV response partially through K(Ca) and Kv channels, but sevoflurane may attenuate the HPV response through other pathways rather than through the currently investigated potassium channels in isolated rabbit lungs.
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Anestésicos por Inhalación/farmacología , Isoflurano/farmacología , Éteres Metílicos/farmacología , Bloqueadores de los Canales de Potasio , Circulación Pulmonar/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Algoritmos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Hipoxia/fisiopatología , Técnicas In Vitro , Conejos , SevofluranoRESUMEN
Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.
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Citrobacter freundii/genética , Mutación del Sistema de Lectura/genética , Sistema de Lectura Ribosómico/genética , Proteínas de Transporte de Membrana/genética , Simportadores , Secuencia de Aminoácidos , Secuencia de Bases , Cationes/metabolismo , Citrobacter freundii/metabolismo , Clonación Molecular , Escherichia coli/genética , Genes Reporteros/genética , Hidrógeno/metabolismo , Melibiosa/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Operón/genética , Mapeo Restrictivo , Salmonella typhimurium , Eliminación de Secuencia/genética , Homología de Secuencia de Aminoácido , Sodio/metabolismo , beta-GalactosidasaRESUMEN
Water forms three-dimensional polymeric structures due to the influence of hydrogen bonds and is fundamentally different from other substances. One of the simplest ways to analyze the structure of water in any system, such as hydration, is to measure the degree of compressibility, which can be determined from the speed of sound, by making use of the physical laws established by Newton and later perfected by Laplace. Although the speed of sound is strongly dependent on the temperature of a liquid, Laplace's equation does not refer to temperature in any of its terms. It is necessary, therefore, to determine the degree of temperature dependency. However, only approximate expressions of a fifth-order polynomial have been reported so far in the literature. In this paper, a universal method for describing the speed of sound from the perspective of physicochemical reaction kinetics is presented. It is shown that the speed of sound U [ms(-1)] changes with temperature T [K] according to a thermodynamically-derived formula given as U= exp(-A/T-BlnT+C) and that the motion and propagation phenomena of sound energy can also be regarded as chemical reactions.
RESUMEN
A gene, designated amyR, coding for a transcriptional activator involved in amylolytic gene expression has been cloned from Aspergillus oryzae by screening for a clone that enabled to reverse the reduced expression of the alpha-amylase gene (amyB) promoter. amyR encodes 604 amino acid residues of a putative DNA-binding protein carrying a zinc binuclear cluster motif (Zn(II)2Cys6) belonging to the GAL4 family of transcription factors. The amyR gene disruptants showed a significant restricted growth on starch medium and produced little of the amylolytic enzymes including alpha-amylase and glucoamylase compared with a non-disruptant, indicating that amyR is a transcriptional activator gene involved in starch/maltose-induced efficient expression of the amylolytic genes in A. oryzae. In addition, sequencing analysis found that amyR, agdA (encoding alpha-glucosidase), and amyA (encoding alpha-amylase), are clustered on a 12-kb DNA fragment of the largest chromosome in A. oryzae, and that amyR is about 1.5 kb upstream of agdA and transcribed in the opposite direction. Furthermore, transcriptional analysis revealed that the amyR gene was expressed in the presence of glucose comparable to the level in the presence of maltose, while the amylolytic genes were transcribed at high levels only in the presence of maltose.
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Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glicósido Hidrolasas/genética , Transactivadores/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Fusión Artificial Génica , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Proteínas Fúngicas/metabolismo , Expresión Génica , Genes Fúngicos , Operón Lac , Datos de Secuencia Molecular , Volumetría , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We investigated the effect of potent inhalation anaesthetics on nitric oxide (NO) concentration measured by the chemiluminescence method. We found that the NO concentration was increasingly underestimated with increasing concentrations of halothane, isoflurane, enflurane and sevoflurane (r2 = 0.918-0.997, P < 0.01). Statistical analysis showed that the four inhalation agents at the same concentration produced a similar error in the measured NO concentration. In the presence of a fixed concentration of sevoflurane (5.0%), isoflurane (5.2%), enflurane (4.5%) or halothane (6.1%), the rate of reduction in the measured NO concentration increased in proportion to the NO concentration (r2 = 0.909-0.982, P < 0.01). No direct chemical interaction between the potent inhalation agents and NO was detected by gas chromatography-mass spectrometry. We conclude that NO concentration can be underestimated when measured by the chemiluminescence method in the presence of potent inhalation agents. This underestimation may result from emission absorption and/or the quenching phenomenon, but is not attributable to a chemical reaction between the inhalation agent and NO.
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Anestésicos por Inhalación/farmacología , Óxido Nítrico/análisis , Análisis de Varianza , Cromatografía de Gases , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Enflurano/farmacología , Éteres/farmacología , Halotano/farmacología , Isoflurano/farmacología , Mediciones Luminiscentes , Espectrometría de MasasRESUMEN
We started a new home infusion therapy system in July 1999. The home infusion therapy system is made up of doctors, nursing stations, and pharmacies in the community. We coordinate these parties before patient discharge from our hospital and support them when the patient needs hospitalization (for example, he or she develops pneumonia). This report discusses past experiences and future issue.
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Servicios de Atención a Domicilio Provisto por Hospital , Terapia de Infusión a Domicilio/tendencias , Internado y Residencia , Terapia de Infusión a Domicilio/normas , HumanosRESUMEN
To reveal the mechanism of the production of acetate by sake yeast (Saccharomyces cerevisiae), the expression of genes encoding aldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and acetyl-CoA hydrolase (ACH), which are related to acetate production, was investigated. Northern blot analysis using total RNA of sake yeast isolated from sake mash revealed that all of the tested genes, ACS1, ACS2, ALD2/3, ALD4, ALD6 and ACH1, were transcribed during sake fermentation. Transcription of ALD2/3 was detected only in the early stage of sake fermentation. A static culture of sake yeast in hyperosmotic media including 1 M sorbitol or 20% glucose resulted in high acetate production and increased transcription of ALD2/3. This is the same result as reported in an aerobic condition, and induction of ALD2/3 seemed to be one reason for high acetate production at high glucose concentration during fermentation. Overexpression of ACS2 resulted in low acetate production both during small-scale sake fermentation and in a static liquid culture. On the other hand, over-expression of ACS1 did not change acetate productivity significantly in a static culture. These results indicate that ALD2/3 and ACS2 play important roles for acetate production during sake fermentation.
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We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.
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Axones/enzimología , Encéfalo/enzimología , Proteínas de Caenorhabditis elegans , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos , Proteínas Reguladoras de la Apoptosis , Autofagia/fisiología , Familia de las Proteínas 8 Relacionadas con la Autofagia , Encéfalo/citología , Células COS , Proteínas Portadoras/metabolismo , Retículo Endoplásmico Liso/enzimología , Expresión Génica/fisiología , Aparato de Golgi/enzimología , Células HeLa , Humanos , Inmunohistoquímica , Proteínas de Microfilamentos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/inmunología , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Homología de Secuencia de Aminoácido , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The UNC-51 serine/threonine kinase of C. elegans plays an essential role in axonal elongation, and unc-51 mutants exhibit uncoordinated movements. We have previously identified mouse and human cDNAs encoding UNC-51-like kinase (ULK1). Here we report the identification and characterization of the second murine member of this kinase family, ULK2. Mouse ULK2 cDNA encodes a putative polypeptide of 1033 aa which has an overall 52% and 33% amino acid identity to ULK1 and UNC-51, respectively. ULKs and UNC-51 share a typical domain structure of an amino-terminal kinase domain, a central proline/serine rich (PS) domain, and a carboxy-terminal (C) domain. Northern blot analysis showed that ULK2 mRNA is widely expressed in adult tissues. In situ hybridization analysis indicated that ULK2 mRNA is ubiquitously localized in premature as well as mature neurons in developing nervous system. ULK2 gene was mapped to mouse chromosome 11B1.3 and rat chromosome 10q23 by FISH. HA-tagged ULK2 expressed in COS7 cells had an apparent molecular size of approximately 150 kDa and was autophosphorylated in vitro. Truncation mutants suggested that the autophosphorylation occurs in the PS domain. Although expression of ULK2 failed to rescue unc-51 mutant of C. elegans, a series of ULK2/UNC-51 chimeric kinases revealed that function of the kinase and PS domains are conserved among species, while the C domain acts in a species-specific manner. These results suggest that ULK2 is involved in a previously uncharacterized signaling pathway in mammalian cells.
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Proteínas de Caenorhabditis elegans , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Caenorhabditis elegans , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Expresión Génica , Ratones , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero , Ratas , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
A Mycoplasma pneumoniae cytadhesin P1 gene with novel nucleotide sequence variation has been identified. Four clinical strains of M. pneumoniae were found to carry this type of P1 gene. This new P1 gene is similar to the known group II P1 genes but possesses novel sequence variation of approximately 300 bp in the RepMP2/3 region. The position of the new variable region is distant from the previously reported variable regions known to differ between group I and II P1 genes. Two sequences closely homologous to this new variable region were found within the repetitive sequences outside the P1 gene of the M. pneumoniae M129 genome. This suggests that the new P1 gene was generated by DNA recombination between repetitive sequences and the P1 gene locus. The finding of this new type of P1 gene supports the hypothesis that the repetitive sequences of the M. pneumoniae genome serve as a reservoir to generate antigenic variation of the cytadhesin P1 gene.
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Adhesinas Bacterianas/genética , Variación Antigénica , Mycoplasma pneumoniae/genética , Recombinación Genética , Adhesinas Bacterianas/inmunología , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Secuencia de Bases , ADN , ADN Bacteriano , Humanos , Datos de Secuencia Molecular , Mycoplasma pneumoniae/inmunología , Neumonía por Mycoplasma/microbiología , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
The chemiluminescent emission reaction dependence on the activity of phagocytosis is well known. However, this method is not used to diagnostically in clinical assessment because the relationship between phagocytizing activity and chemiluminescent intensity has not been clearly established. Therefore, we attempted to analyze quantitatively the chemiluminescent emission curve by the phagocytosis of leukocytes. Mathematical assessment of the emission curve with respect to time was performed by fitting the curve to several regression models using the unweighed non-linear least squares method. A triple logarithmic normal distribution model provided a reasonable goodness of fit to the measured emission curve. The first component, about 5% of the calculated total counts, was assumed to arise from monocytes activity, the second component, about 20% from eosinocytes activity and the third component, up to 75%, from neutrophils activity. This method seems promising as a means for assaying whole blood without the need for pretreatment and for the providing a valid index that is independent of the technical differences between laboratories.
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Leucocitos/inmunología , Mediciones Luminiscentes , Consumo de Oxígeno , Fagocitosis/inmunología , Interpretación Estadística de Datos , HEPES , Humanos , Luminol , Modelos Biológicos , Monocitos/inmunología , Neutrófilos/inmunología , ZimosanRESUMEN
Two-step polymerase chain reaction (PCR) with primers designated against 16S rRNA gene of Mycoplasma pneumoniae for diagnosis of infection was evaluated in comparison with the conventional single-step PCR and culture methods. The two-step PCR method showed specific amplification of M. pneumoniae DNA and higher sensitivity (1.5 fg/assay) than the single-step PCR method. With the two-step PCR method, 76 of 322 throat swabs (23.6%) from patients with acute respiratory complaints gave positive results whereas 20.2% were positive in the culture method. Seven of 13 samples which were negative in the single-step PCR method but positive in either serological or the culture method showed positive results by the two-step PCR method. In addition, 5 samples which were weakly positive in the single-step PCR method showed distinctly positive results in the two-step PCR. These results indicate that the two-step PCR method is a useful tool for detection of M. pneumoniae in clinical specimens, although it requires a relatively sophisticated in technique.
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Mycoplasma pneumoniae/aislamiento & purificación , Faringe/microbiología , Reacción en Cadena de la Polimerasa/métodos , HumanosRESUMEN
We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1.7 Mb to 3.5 Mb and total genome size was estimated to be 9.5 Mb to 9.8 Mb; however, chromosome-length polymorphisms existed among four strains. Thirteen cloned genes isolated from strain GTS115 were assigned to the separated chromosomes, revealing that different hybridization patterns were observed in the AOX2 and URA3 genes among strains. P. pastoris is frequently used as an efficient host for heterologous gene expressions. We analysed chromosomal stability of strain GTS115-derived recombinant cell expressing human serum albumin during serial cultivation under the condition of vegetative and non-selective growth. No chromosomal rearrangements were observed and the expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expression would be carried out even in large-scale fermentation.