RESUMEN

Consumption of seafood contaminated with ciguatoxins (CTXs) leads to a foodborne disease known as ciguatera. Primary producers of CTXs are epibenthic dinoflagellates of the genera Gambierdiscus and Fukuyoa. In this study, thirteen Gambierdiscus and Fukuyoa strains were cultured, harvested at exponential phase, and CTXs were extracted with an implemented rapid protocol. Microalgal extracts were obtained from pellets with a low cell abundance (20,000 cell/mL) and were then analyzed with magnetic bead (MB)-based immunosensing tools (colorimetric immunoassay and electrochemical immunosensor). It is the first time that these approaches are used to screen Gambierdiscus and Fukuyoa strains, providing not only a global indication of the presence of CTXs, but also the ability to discriminate between two series of congeners (CTX1B and CTX3C). Analysis of the microalgal extracts revealed the presence of CTXs in 11 out of 13 strains and provided new information about Gambierdiscus and Fukuyoa toxin profiles. The use of immunosensing tools in the analysis of microalgal extracts facilitates the elucidation of further knowledge regarding these dinoflagellate genera and can contribute to improved ciguatera risk assessment and management.

Asunto(s)

Ciguatoxinas/aislamiento & purificación , Colorimetría/métodos , Dinoflagelados/química , Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Ciguatoxinas/clasificación , Especificidad de la Especie

RESUMEN

Agaricus blazei Murill is an edible fungus used in traditional medicine, which has various well-documented medicinal properties. In the present study, we investigated the effects of hemicellulase-derived mycelia extract (Agaricus blazei fraction H: ABH) on the immune system. First, we examined the cytokine-inducing activity of ABH on human peripheral mononuclear cells (PBMC). The results indicated that ABH induced expression of IL-12, a cytokine known to be a critical regulator of cellular immune responses. Flow cytometric analysis demonstrated the induction of IL-12 production by the CD14-positive cell population, consisting of monocytes/macrophages (Mo/Mphi). Furthermore, the elimination of Mo/Mphi attenuated IL-12 production in PBMC. ABH-induced IL-12 production was inhibited by anti-CD14 and anti-TLR4 antibodies but not by anti-TLR2 antibody. The activity of ABH was not inhibited by polymyxin B, while the activity of lipopolysaccharide used as a reference was inhibited. Oral administration of ABH enhanced natural killer (NK) activity in the spleen. These findings suggest that ABH activated Mo/Mphi in a manner dependent on CD14/TLR4 and NK activity.

RESUMEN

More than 20,000 people suffer annually from ciguatera seafood poisoning in subtropical and tropical regions. The extremely low content of the causative neurotoxins, designated as ciguatoxins, in fish has hampered the isolation, detailed biological studies, and preparation of anti-ciguatoxin antibodies for detecting these toxins. The large (3 nanometers long) and complicated molecular structure of ciguatoxins has impeded chemists from completing their total synthesis. Our highly convergent strategic approach featuring the chemoselective ring-closing metathesis reaction as a key tactic has enabled the total synthesis of ciguatoxin CTX3C, which will provide a practical supply for further studies.

Asunto(s)

Ciguatoxinas/síntesis química , Dinoflagelados/química , Animales , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Ciguatoxinas/química , Ciguatoxinas/inmunología , Ciguatoxinas/toxicidad , Dicroismo Circular , Isomerismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Alimentos Marinos/parasitología , Alimentos Marinos/envenenamiento

Asunto(s)

Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Daño del ADN , ADN/efectos de los fármacos , Compuestos Epoxi/síntesis química , Compuestos Epoxi/farmacología , Naftalenos/síntesis química , Naftalenos/farmacología , ADN/química , Enediinos , Estereoisomerismo , Especificidad por Sustrato

RESUMEN

[reaction: see text]. A protected version of the northern part of TMC-95A, a potent and selective proteasome inhibitor, was synthesized with full stereochemical control. Highlights of this synthesis include (i) a (Z)-selective Mizoroki-Heck reaction to construct the oxyindole portion, (ii) a diastereoselective epoxidation, (iii) a 6-endo selective epoxide opening by Boc carbonyl group to establish the stereochemistry of C6, and (iv) a 1,3-elimination reaction of the L-allo-threonine derivative under Mitsunobu conditions to afford the (Z)-1-propenylamine.

Asunto(s)

Péptidos Cíclicos/síntesis química , Inhibidores de Proteasas/síntesis química , Cisteína Endopeptidasas , Conformación Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal

RESUMEN

C-1027 is one of the most potent antitumor antibiotic chromoproteins, and is a 1:1 complex of an enediyne chromophore having DNA-cleaving ability and a carrier apoprotein. The three-dimensional solution structures of the 110 residue (10.5 kDa) C-1027 apoprotein and its complex with the aromatized chromophore have been determined separately by homonuclear two-dimensional nuclear magnetic resonance methods. The apoprotein is mainly composed of three antiparallel beta-sheets: four-stranded beta-sheet (43-45, 52-54; 30-38; 92-94; 104-106), three-stranded beta-sheet (4-6; 17-22; 61-66), and two-stranded beta-sheet (70-72; 83-85). The overall structure of the apoprotein is very similar to those of other chromoprotein apoproteins, such as neocarzinostatin and kedarcidin. A hydrophobic pocket with approximate dimensions of 14 A x 12 A x 8 A is formed by the four-stranded beta-sheet and the three loops (39-42; 75-79; 97-100). The holoprotein (complex form with the aromatized chromophore) structure reveals that the aromatized chromophore is bound to the hydrophobic pocket found in the apoprotein. The benzodihydropentalene core of the chromophore is located in the center of the pocket and other substituents (beta-tyrosine, benzoxazine, and aminosugar moieties) are arranged around the core. Major binding interactions between the apoprotein and the chromophore are likely the hydrophobic contacts between the core of the chromophore and the hydrophobic side-chains of the pocket-forming residues, which is supplemented by salt bridges and/or hydrogen bonds. Based on the holoprotein structure, we propose possible mechanisms for the stabilization and the release of chromophore by the apoprotein.

Asunto(s)

Alquinos/química , Alquinos/metabolismo , Aminoglicósidos , Antibacterianos/química , Antibacterianos/metabolismo , Cicloparafinas/química , Cicloparafinas/metabolismo , Naftalenos/química , Naftalenos/metabolismo , Péptidos , Actinomycetales/química , Secuencia de Aminoácidos , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Sitios de Unión , Ciclización , Enediinos , Enlace de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Soluciones , Solventes , Estereoisomerismo

RESUMEN

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Ciguatoxinas/metabolismo , Epítopos/metabolismo , Albúmina Sérica Bovina/metabolismo , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos/fisiología , Ciguatoxinas/síntesis química , Desoxiglucosa/análogos & derivados , Desoxiglucosa/síntesis química , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Conformación Molecular , Albúmina Sérica Bovina/química , Resonancia por Plasmón de Superficie

Asunto(s)

Antibióticos Antineoplásicos/síntesis química , Compuestos Epoxi/química , Naftalenos/química , Streptomyces/química , Antibióticos Antineoplásicos/química , ADN/metabolismo , Enediinos , Compuestos Epoxi/síntesis química , Estructura Molecular , Naftalenos/síntesis química

RESUMEN

Twenty-five patients who received home medical treatment were reviewed. There were 11 males and 14 females, with a mean age of 77 years. In an orthopedic clinic, it is difficult to secure the time for to visit a patient's home. Another problem for doctor is that the medical fee for home treatment is very low. To promote home treatment by orthopedists, it is important to secure the time to visit a patient's home, to improve the fees for home treatment, and to establish a connection with a visiting nurse care station and visiting rehabilitation workers.

Asunto(s)

Trastornos Cerebrovasculares/terapia , Fracturas por Estrés/terapia , Servicios de Atención de Salud a Domicilio , Ortopedia , Anciano , Anciano de 80 o más Años , Trastornos Cerebrovasculares/rehabilitación , Enfermería en Salud Comunitaria , Femenino , Fracturas por Estrés/rehabilitación , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

Human FcepsilonRII/CD23 is an approximately 45 kDa type II transmembrane glycoprotein belonging to the C-type animal-lectin family, and has two isoforms (a and b) that only differ in their intracytoplasmic tails. We previously found that in several human and mouse cell lines there were two additional CD23 transcripts (a' and b') lacking the exon 3 that encodes the entire transmembrane segment and a part of cytoplasmic tails. In this study, we analyzed the putative CD23a' and CD23b' products at protein levels and characterized with rabbit polyclonal antibodies against novel amino-acid sequences of the putative CD23a' and CD23b' molecules (anti-CD23a' Ab, anti-CD23b' Ab). Western blots in COS cells transfected with CD23a' or CD23b' cDNA as well as in vitro translation assays showed that the a' and b' CD23 transcripts were translated to about 40 kDa molecules. These 40 kDa molecules were also recognized by a polyclonal antibody against 25 kDa soluble fragment of human CD23. We also found that human cells having mRNAs for CD23a' and CD23b' expressed protein products recognized specifically by anti-CD23a' or anti-CD23b' Ab, respectively. In addition, the CD23a' and CD23b' molecules in transfected COS cells were resistant to Endo H(f) and PNGase F, although these truncated forms as well as the membrane-associated forms had an asparagine residue responsible for the N-linked glycosylation. Taken together, our results show that the a' and b' CD23 transcripts are expressed and translated in human lymphoid cells and that their translated products are retained in the cytoplasm where they might play an unique regulatory role in the expression of the full-length CD23 on the cell surface.

Asunto(s)

Receptores de IgE/química , Receptores de IgE/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Secuencia de Bases , Células COS , Línea Celular , Cartilla de ADN/genética , Glicosilación , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Biosíntesis de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Receptores de IgE/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

We created a new imaging technique that detects and emphasizes turbulence, which is a characteristic of blood flow in hepatocellular carcinoma. We devised two indices that determine a characteristic tumor flow, the bi-directional and low-peak indices. In the phantom study, both indices of turbulence caused by a stenosis were much higher. In the clinical study, both indices were significantly higher in tumors than in the portal vein or hepatic vein. A turbulent blood flow was detected in 77% of tumors, whereas such detection seldom occurred in the portal or hepatic vein. This technique has the potential to distinguish turbulence in hepatocellular carcinoma.

Asunto(s)

Velocidad del Flujo Sanguíneo/fisiología , Carcinoma Hepatocelular/irrigación sanguínea , Hemorreología , Neoplasias Hepáticas/irrigación sanguínea , Ultrasonografía Doppler en Color , Biopsia , Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/patología , Estudios de Evaluación como Asunto , Venas Hepáticas/diagnóstico por imagen , Venas Hepáticas/fisiopatología , Humanos , Procesamiento de Imagen Asistido por Computador , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/patología , Modelos Anatómicos , Vena Porta/diagnóstico por imagen , Vena Porta/fisiopatología , Flujo Sanguíneo Regional , Ultrasonografía Doppler en Color/métodos

RESUMEN

To produce a thrombi-targeting plasminogen activator, we expressed a fused gene that contains a modified pre-sequence of Mucor pussilus rennin (MPR) followed by a chimeric gene of single-chain urokinase-type plasminogen activator (scu-PA)::annexin V (AV). The fused gene was ligated into an integrative vector, under the control of the alcohol oxidase 1 (AOX1) promoter (p), and transformed into Pichia pastoris. Transformants were monitored for the secretion of fibrinolytic activity. The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per ml of culture medium under optimal conditions. It contained three tandem copies of the full-size vector disruptively integrated into the AOX1 sequence. Western blot analysis revealed that the secreted chimera was highly susceptible to proteolysis. Addition of excess amino acids (aa) to the culture medium minimized the degree of proteolysis. Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium. The former was the intact form consisting of a single-chain and showing full enzyme activity after activation by plasmin. The latter was an enzymatically processed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation. Both chimeras exhibited calcium-dependent phospholipid (PL)-binding affinities similar to the parent AV.

Asunto(s)

Anexina A5/genética , Pichia/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Secuencia de Aminoácidos , Anexina A5/aislamiento & purificación , Anexina A5/metabolismo , Secuencia de Bases , Membrana Celular/metabolismo , ADN Recombinante , Fibrinólisis , Vectores Genéticos , Datos de Secuencia Molecular , Mucor , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transformación Genética , Activador de Plasminógeno de Tipo Uroquinasa/aislamiento & purificación , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo

RESUMEN

The ectodomain of the alpha subunit of the human high-affinity receptor for IgE (Fc epsilon RI) has been engineered as a recombinant soluble protein (sFc epsilon RI alpha) and purified in large quantity from the supernatant of transfected mammalian cells. The sFc epsilon RI alpha was apparently heterogeneous in terms of molecular weight (40 to 60 kd), and this heterogeneity was largely due to N-linked carbohydrates on the molecule. The amino terminal sequence was homogeneous and identical to the sequence predicted from the complementary DNA sequence. The amino acid composition of the sFc epsilon RI alpha was in agreement with the values expected from the cDNA sequence. The sFc epsilon RI alpha formed a complex with IgE, and gel filtration analyses of the complex of the sFc epsilon RI alpha and human IgE supported the idea that the stoichiometry of the IgE binding was 1:1. Passive anaphylactic shock in mice was suppressed when the sFc epsilon RI alpha was intravenously injected after sensitization with anti-trinitrophenyl IgE and challenged with the appropriate antigen. In this model the sFc epsilon RI alpha was effective when administered 48 hours before the antigen challenge, but not 24 hours before the challenge. These results suggest that the sFc epsilon RI alpha facilitated the dissociation of IgE from Fc epsilon RI on mast cells in vivo and suppressed type I allergic reactions.

Asunto(s)

Anafilaxia/prevención & control , Receptores de IgE/genética , Receptores de IgE/fisiología , Proteínas Recombinantes/uso terapéutico , Secuencia de Aminoácidos , Aminoácidos/análisis , Anafilaxia/inmunología , Anafilaxia/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina E/química , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Unión Proteica/inmunología , Receptores de IgE/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Solubilidad , Transfección

RESUMEN

Caryoynencins (1) are antibiotics isolated from liquid cultures of a plant pathogen, Pseudomonas caryophylli, and are unstable C18 carboxylic acids with a conjugated dienetetrayne structure. Enyne analogs of caryoynencins were synthesized from monosilylated 1,3-butadiyne 2 (n = 2), 1,3,5-hexatriyne 2 (n = 3), and 1,3,5,7-octatetrayne 2 (n = 4) by alkynyl metal addition to 2,4-hexadienal (3) followed by allylic rearrangement and deprotection. Tetraynol 5 (n = 4) thus obtained was resolved by enzyme reactions. The conjugated dienetetrayne compounds are mixtures of 3E,5E- and 3E,5Z-isomers, which equilibrate by room light. 13C-NMR chemical shifts of polyynes obey simple rules, which can be used for signal assignments. Antimicrobial activities of conjugated enynes and related compounds were examined. The tetrayne analog 6 (n = 4) possesses potent antibacterial and antifungal activities, while triyne and diyne analogs 6 (n = 3 and 2) are less active. Chirality does not affect the activities. An isomeric enyne compound, 2,4-tetradecadiene-7,9,11,13-tetrayn-6-ol (8), showed potent activity against Tricophyton.

Asunto(s)

Alquinos/farmacología , Antibacterianos/farmacología , Antifúngicos/farmacología , Pseudomonas/química , Alquinos/síntesis química , Alquinos/química , Antibacterianos/síntesis química , Antibacterianos/química , Antifúngicos/síntesis química , Antifúngicos/química , Bacterias/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Hongos Mitospóricos/efectos de los fármacos , Mucor/efectos de los fármacos , Relación Estructura-Actividad

RESUMEN

A new method has been developed for measuring the volume flow rate of blood flowing through large vessels or outflow tracts of the heart. In this article we describe the principle of a method that can reduce the dependence of the Doppler angle of flow measurement by setting the sample points along a line to which every ultrasound beam is perpendicular. To evaluate the accuracy of this method, flow phantom experiments were made for both steady and pulsatile flows. The volume flow rate measured by this method agrees well with that observed by an ultrasound flowmeter (r = 0.99) when the vessel diameter is large (25 mm). However, this method overestimates by 40% when the vessel diameter is small (8 mm). To make this method applicable to small vessels, an improvement in the lateral resolution of Doppler measurement is necessary. It has been concluded that this method can be used to measure the cardiac output or volume flow rates in large vessels.

Asunto(s)

Gasto Cardíaco , Ecocardiografía Doppler/métodos , Algoritmos , Velocidad del Flujo Sanguíneo , Volumen Cardíaco , Ecocardiografía Doppler/instrumentación , Ecocardiografía Doppler en Color , Ecocardiografía Doppler de Pulso , Predicción , Hemorreología , Humanos , Aumento de la Imagen/métodos , Modelos Cardiovasculares , Modelos Estructurales , Modelos Teóricos , Flujo Pulsátil , Volumen Sistólico

Asunto(s)

Cinostatina/química , ADN/química , Fotoquímica

RESUMEN

OBJECTIVES: This study was performed to assess a new indicator of regional left ventricular contraction determined by a two-dimensional tissue Doppler imaging technique. BACKGROUND: Recent studies have demonstrated that instantaneous tissue motion velocity can be noninvasively assessed by tissue Doppler imaging. However, quantitative assessment of regional left ventricular contraction is still difficult because of the effects of the Doppler angle of incidence and parallel motion of the whole heart. METHODS: We assessed left ventricular wall motion in 11 normal subjects, 14 patients with an old myocardial infarction (anteroseptal in 7, posterior in 7) and 8 patients with dilated cardiomyopathy. Tissue Doppler velocity was corrected by the Doppler angle of incidence after the hypothetical center of contraction was set. Subsequently, the myocardial velocity gradient between the endocardium and epicardium was determined from the velocity profile along each radial line from the center of contraction by using least squares linear regression. RESULTS: In normal subjects, peak myocardial velocity gradient was lower in the anteroseptal wall (mean [+/- SD] 1.69 +/- 0.53 s-1) than in the posterior wall (3.28 +/- 0.67 s-1, p < 0.01). Myocardial velocity gradient in the infarct regions was significantly lower (anteroseptal 0.58 +/- 0.41 s-1, p < 0.05; posterior 0.17 +/- 0.27 s-1, p < 0.01) than that in normal subjects as well as that in the corresponding noninfarct regions (2.84 +/- 0.37 s-1 and 1.48 +/- 0.25 s-1, p < 0.01, respectively). In patients with dilated cardiomyopathy, myocardial velocity gradient was generally lower (anteroseptal 0.72 +/- 0.59 s-1; posterior 0.93 +/- 0.67 s-1) than that in normal subjects (p < 0.01). CONCLUSIONS: These results demonstrate that regional left ventricular contraction can be quantitatively assessed by the myocardial velocity gradient derived from two-dimensional tissue Doppler imaging. We suggest that myocardial velocity gradient has potential for the quantitative assessment of regional left ventricular contraction abnormalities in patients.

Asunto(s)

Cardiomiopatía Dilatada/fisiopatología , Ecocardiografía Doppler , Contracción Miocárdica/fisiología , Infarto del Miocardio/fisiopatología , Adulto , Anciano , Cardiomiopatía Dilatada/diagnóstico por imagen , Estudios de Evaluación como Asunto , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Análisis de los Mínimos Cuadrados , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico por imagen , Función Ventricular