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PURPOSE OF REVIEW: Fecal microbial transplantation (FMT) has become established as an effective therapeutic modality in the treatment of antibiotic-refractory recurrent Clostridium difficile colitis. A number of formulations and methods of delivery of FMT are currently available, each with distinct advantages. This review aims to review donor and patient selection for FMT as well as procedural aspects of FMT to help guide clinical practice. RECENT FINDINGS: FMT can be obtained in fresh, frozen, lyophilized, and capsule-based formulations for delivery by oral ingestion, nasoenteric tube, colonoscopy, or enema (depending on the formulation used). Choosing the optimal method relies heavily on patient-related factors, including underlying pathology and severity of illness. As potential applications for FMT expand, careful donor screening and patient selection are critical to minimizing risk to patients and physicians. FMT represents an excellent therapeutic option for treatment of recurrent Clostridium difficile colitis and holds promise as a possible treatment modality in a variety of other conditions. The wide array of delivery methods allows for its application in various disease states in both the inpatient and outpatient setting.
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Trasplante de Microbiota Fecal/métodos , Selección de Donante , Enterocolitis Seudomembranosa/terapia , Humanos , Selección de PacienteRESUMEN
Despite its legal and scientific failings, the "intelligent design" (ID) movement has been a public relations success story in the United States. By first creating doubts about the adequacy of evolution to account for the complexity of life, the ID movement has invoked the values of "fairness" and "openness" to argue for inclusion in the classroom and curriculum. In this way, it has attempted to lay claim to the very principles of critical analysis and open discussion at the heart of the scientific enterprise, leaving many researchers in doubt as to how to respond to these challenges. Specific case studies, including the blood-clotting cascade and data from the human genome, show how scientists can have a leading role in deconstructing the arguments advanced in favor of ID. The key to this strategy is remarkably simple and was at the heart of the landmark 2005 Kitzmiller v. Dover trial on ID. It is for researchers to take the claims made by ID proponents seriously, and then to follow them to their logical scientific conclusions. When this is done effectively, the hypothesis of "design" can be publicly falsified in ways that are understandable to laypeople and decision makers in education.
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Evolución Biológica , Religión y Ciencia , Animales , Coagulación Sanguínea/genética , Cromosomas Humanos/genética , Curriculum , Fósiles , Genoma Humano , Humanos , Inteligencia , Ciencia/educación , Ciencia/legislación & jurisprudencia , Estados UnidosRESUMEN
Nonquadratic regularizers, in particular the l(1) norm regularizer can yield sparse solutions that generalize well. In this work we propose the generalized subspace information criterion (GSIC) that allows to predict the generalization error for this useful family of regularizers. We show that under some technical assumptions GSIC is an asymptotically unbiased estimator of the generalization error. GSIC is demonstrated to have a good performance in experiments with the l(1) norm regularizer as we compare with the network information criterion (NIC) and cross- validation in relatively large sample cases. However in the small sample case, GSIC tends to fail to capture the optimal model due to its large variance. Therefore, also a biased version of GSIC is introduced,which achieves reliable model selection in the relevant and challenging scenario of high-dimensional data and few samples.
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Trisomy 6 and trisomy 6 mosaicism were found in chorionic villi cell culture and short term incubation in a prenatal diagnosis at 12 weeks of gestation in a pregnancy with a growth retarded fetus showing nuchal translucency. The child was born in the 25th gestational week with a number of malformations including heart defects, deep-set ears, cleft right hand, cutaneous syndactylies, and overlapping toes of irregular shape and length. Trisomy 6 was not found in peripheral blood lymphocytes but was confirmed in umbilical cord fibroblasts. Currently, at the age of 2-3/4 years, the development of the child is relatively normal despite considerable growth delay. At the age of two years, she developed a papular erythema clinically suggestive of epidermal nevi. Cytogenetic analysis of fibroblast cultures derived from skin from a right hand finger and the inguinal area confirmed the presence of a trisomy 6 mosaicism. This is the first observation of a liveborn with trisomy 6 mosaicism.
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Cromosomas Humanos Par 6 , Mosaicismo/genética , Trisomía/genética , Preescolar , Femenino , HumanosRESUMEN
Historically, ensuring the due process rights of deaf defendants has been a problematic issue in the criminal justice system (McAlister, 1994; Smith, 1994; Vernon & Coley, 1978; Vernon & Greenburg, 1996; Vernon & Miller, in press; Vernon & Raifman, 1997; Whalen, 1981; Wood, 1984). Inadequate communication can radically affect a deaf defendant's interactions in the courtroom. Pursuant to the concepts of fairness enshrined in the U.S. Constitution and the specific statutory language contained in federal and state laws, the courts must provide equal access for deaf defendants (Berko, 1992; Gallie & Smith, 2000; McCoy, 1992; Simon, 1994; Vernon & Raifman, 1997). It is the responsibility of the court to ensure that the appropriate accommodation is provided in the language most readily understood by the defendant.When adjudicating a deaf criminal defendant, courts must make certain that the defendant has equal access to various due process activities, such as assisting counsel in the development of a defense, deciding whether to testify, deciding which plea to enter, understanding the charges, understanding one's position as defendant, and comprehending the role of the defense and prosecuting attorneys, and judge (Berko, 1994; King, 1990; Simon, 1994; Smith, 1994; Vernon & Coley, 1978; Vernon & Miller, in press; Vernon, Raifman, & Greenberg, 1996).However, complex linguistic issues that impinge on adjudicative competence are present in some deaf defendants (Vernon & Miller, in press; Vernon & Raifman, 1997). Adjudicative competence refers to an individual's ability to adequately comprehend and participate in legal proceedings and due process activities. When diverse language use is an issue, a deaf defendant's ability to participate in proceedings can be established by the court using the modern test of adjudicative competence (Dusky v. U.S., 1960). This test examines a defendant's state of mind at the time of trial rather than at the time of the offense in terms of these factors: a defendant's capacity to participate, reasonable understanding of the proceedings, and level of cognitive functioning, irrespective of any mental disorder. This article will outline linguistic barriers to due process for deaf defendants.
RESUMEN
Historically, the provision of sign language interpreters to deaf suspects, defendants, and offenders has been a problematic issue in the criminal justice system. Inconsistency in the provision of interpreter services results largely from the ignorance of criminal justice professionals regarding deaf people's communication needs and accommodation options. Through analysis of 22 post-Americans with Disabilities Act cases and a survey of 46 professional sign language interpreters working in criminal justice settings, the present study considered access issues concerning sign language interpreters in law enforcement, courtrooms, and correctional settings. Recommendations to increase the accessibility of interpreting services include providing ongoing awareness training to criminal justice personnel, developing training programs for deaf legal advocates, and continuing access studies.
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Derecho Penal/legislación & jurisprudencia , Sordera , Lengua de Signos , Traducción , HumanosRESUMEN
It has been reported that 14C-labeled methyl-n-amyl ketone (MAK, 2-heptanone) is able to bind spontaneously, in vitro, to isolated rat liver DNA to the extent of 400 pmol/mg DNA; and that 14C-MAK, when given by gavage to female Fischer 344 rats, resulted in HPLC chromatograms of isolated, hydrolyzed liver DNA in which some radiolabel was not associated with the four normal DNA bases dA, dT, dC, and dG. The present studies were undertaken to re-examine the hypothesis that MAK is able to bind to rat liver DNA. In the in vitro study, liver nuclear DNA was incubated with [2-14C]-labeled MAK (25 mCi/mmol) in the absence, or in the presence of rat liver microsomes, precipitated, washed free of unbound MAK, and counted by scintillation spectrometry. No binding to DNA by MAK was detectable. In the in vivo study, groups of five female F344 rats were exposed by inhalation to 0, 80, 400, or 1000 ppm MAK for 6 h/day for 10 days. DNA was purified from the liver nuclei of the 0 and 1000 ppm dosed animals, and 32P-postlabeling techniques were used to assay for adducts. No DNA adducts were detected using these techniques. It was concluded that MAK lacks the ability to bind to rat liver DNA in vitro and in vivo.
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Aductos de ADN/metabolismo , Cetonas/metabolismo , Hígado/metabolismo , Animales , Autorradiografía , Cromatografía en Capa Delgada , ADN/aislamiento & purificación , Femenino , Hígado/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Radioisótopos de Fósforo , Ratas , Ratas Endogámicas F344RESUMEN
OBJECTIVE: To report the natural history and clinical significance of indocyanine green (ICG) angiographic hot spots in age-related macular degeneration (ARMD) after laser photocoagulation. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Two hundred thirty consecutive patients with exudative ARMD who underwent krypton laser treatment between March 1993 and December 1996. INTERVENTION: Krypton laser photocoagulation was performed on all patients. Digital videoangiograms, including both fluorescein angiography and ICG angiography, were obtained at 2 weeks, 4 weeks, 6 weeks, and 3 months postlaser treatment on each patient and repeated if clinical changes were noted. MAIN OUTCOME MEASURES: Detection by ICG of hyperfluorescent spots (hot spots) within the hypofluorescent laser-treated area. RESULTS: Forty patients (18%) developed ICG hot spots 2 weeks after laser treatment. The hot spots disappeared spontaneously without recurrent choroidal neovascular (CNV) membrane in 31 patients (78%). Recurrent CNV was discovered at the hot spot in four patients and away from the hot spot in five patients. CONCLUSION: The postlaser ICG angiographic hot spot is not rare. These spots tend to disappear spontaneously and do not necessarily represent a recurrent CNV. The presence of a hot spot on ICG angiography is not an indication for immediate laser retreatment.
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Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/cirugía , Angiografía con Fluoresceína/métodos , Verde de Indocianina , Coagulación con Láser , Degeneración Macular/complicaciones , Anciano , Anciano de 80 o más Años , Neovascularización Coroidal/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
The SecYEG complex is a major component of the protein translocation apparatus in the cytoplasmic membrane of bacteria. We have purified a translocationally active complex of the two subunits, SecY and SecE, from Bacillus subtilis. As demonstrated by electron microscopy, SecY/E forms ring structures in detergent solution and in intact lipid bilayers, often with a quasi-pentagonal appearance in projection. The particles represent oligomeric assemblies of the SecY/E complex and are similar to those formed by the eukaryotic Sec61p complex. We propose that these SecY/E rings represent protein-conducting channels and that the two essential membrane components SecY and SecE are sufficient for their formation.
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Proteínas Bacterianas/química , Proteínas de Escherichia coli , Proteínas de la Membrana/química , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Transporte Biológico Activo , Detergentes , Escherichia coli/genética , Expresión Génica , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Conformación Proteica , Proteolípidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Translocación SEC , SolucionesRESUMEN
The multisubunit IkappaB kinase (IKK) catalyzes the signal-inducible phosphorylation of N-terminal serines of IkappaB. This phosphorylation is the key step in regulating the subsequent ubiquitination and proteolysis of IkappaB, which then releases NF-kappaB to promote gene transcription. As measured by 33P incorporation into a GST-IkappaB alpha fusion protein, varying both the concentration of GST-IkappaB alpha and [gamma-33P]ATP resulted in a kinetic pattern consistent with a random, sequential binding mechanism. Values of 55 nM and 7 microM were obtained for the dissociation constants of GST-IkappaB alpha and ATP, respectively. The value of alpha, a factor by which binding of one substrate changes the dissociation constant for the other substrate, was determined to be 0.11. This indicates that the two substrates bind in a cooperative fashion. Peptides corresponding to either amino acids 26-42 (N-terminal peptide) or amino acids 279-303 (C-terminal peptide) of IkappaB alpha inhibited the IKK-catalyzed phosphorylation of GST-IkappaB alpha; the C-terminal peptide, unexpectedly, was more potent. The inhibition by the C-terminal peptide was competitive with respect to GST-IkappaB alpha and mixed with respect to ATP, which verified the sequential binding mechanism. The C-terminal peptide was also a substrate for the enzyme, and a dissociation constant of 2.9-6.2 microM was obtained. Additionally, the N-terminal peptide was a substrate (Km = 140 microM). Competitive inhibition of the IKK-catalyzed phosphorylation of the C-terminal peptide by the N-terminal peptide indicated that the peptides are phosphorylated by the same active site. Surprisingly, the presence of the C-terminal peptide greatly accelerated the rate of phosphorylation of the N-terminal peptide as represented by a 160-fold increase in the apparent second-order rate constant (kcat/Km). These results are consistent with an allosteric site present within IKK that recognizes the C terminus of IkappaB alpha and activates the enzyme. This previously unobserved interaction with the C terminus may represent an important mechanism by which the enzyme recognizes and phosphorylates IkappaB.
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Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Fragmentos de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Catálisis , Activación Enzimática , Células HeLa , Humanos , Quinasa I-kappa B , Cinética , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
Interfacial binding affinities and capacities of lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein A-I (apoA-I) for surfaces of different phosphatidylcholine (PC) composition, cholesterol content, and apolipoprotein content were measured with a vesicle model system. Native polyacrylamide gel electrophoresis was used to separate free protein from vesicle-bound protein. ApoA-I was isolated from human plasma and radiolabeled with iodine, whereas radiolabeled LCAT was purified from the media of Chinese hamster ovary cells that were transfected with human LCAT cDNA and incubated in the presence of [35S] cysteine and methionine. Bound and free radiolabeled LCAT and apoA-I were quantified by phosphorimage analysis. ApoA-I binding was not influenced by cholesterol content (14 mole%) but was influenced by the PC fatty acyl composition of the vesicle. PC species containing long chain, polyunsaturated fatty acids (PUFA) in the sn-2 position resulted in increased binding affinity (Kd = 75-177 nM) but reduced capacity (0.1-0.3 apoA-I/ 1000 PC) in comparison to sn-1 palmitoyl, sn-2 oleoyl PC (POPC, 750 nM and 1.4 apoA-I/1000 PC). LCAT binding affinity to POPC (2190 nM) was stronger in the presence of cholesterol (530 nM), and LCAT binding capacity was reduced (2.63 and 0.6 molecules LCAT/1000 PC, respectively). In comparison to POPC, LCAT binding affinity to sn-1 palmitoyl, sn-2 arachidonyl PC was stronger (611 nM) and binding capacity was reduced (0.7 LCAT/1000 PC). LCAT binding affinity and capacity to sn-1 palmitoyl, sn-2 eicosapentaneoyl PC (2041 nM, and 2.5 LCAT/1000 PC) were similar to those observed for POPC. We conclude that vesicle surface PC fatty acyl composition and cholesterol content significantly influence LCAT and apoA-I interfacial binding and therefore may alter LCAT enzymatic activity.
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Apolipoproteína A-I/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfatidilcolinas/química , Animales , Apolipoproteína A-I/aislamiento & purificación , Unión Competitiva , Células CHO , Colesterol/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Humanos , Liposomas/química , Liposomas/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolinas/metabolismo , Radioisótopos de Azufre , Propiedades de Superficie , TransfecciónRESUMEN
To determine whether treatment with L-arginine or superoxide dismutase (SOD) would prove effective in reducing cerebral hypoperfusion after traumatic brain injury (TBI), we measured cerebral blood flow (CBF) using laser Doppler flowmetry (LDF) in rats treated before or after moderate (2.2 atm) fluid-percussion (FP) TBI. Rats were anesthetized with isoflurane and prepared for midline FP TBI and then for LDF by thinning the calvaria using an air-cooled drill. Rats were then randomly assigned to receive sham injury, sham injury plus L-arginine (100 mg/kg, 5 min after sham TBI), TBI plus 0.9% NaCl, TBI plus L-arginine (100 mg/kg, 5 min post-TBI), TBI plus SOD (24,000 U/kg pre-TBI + 1600 units/kg/min for 15 min after TBI), or TBI plus SOD and L-arginine. A second group of rats received TBI plus saline, L-, or D-arginine (100 mg/kg, 5 min after-TBI). After treatment and TBI or sham injury, CBF was measured continuously using LDF for 2 h and CBF was expressed as a percent of the preinjury baseline for 2 h after TBI. Rats treated with saline or D-arginine exhibited significant reductions in CBF that persisted throughout the monitoring period. Rats treated with L-arginine alone or in combination with SOD exhibited no decreases in CBF after TBI. CBF in the SOD-treated group decreased significantly within 15 min after TBI but returned to baseline levels by 45 min after TBI. These studies indicate that L-arginine but not D-arginine administered after TBI prevents posttraumatic hypoperfusion and that pretreatment with SOD will restore CBF after a brief period of hypoperfusion.
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Arginina/uso terapéutico , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/fisiopatología , Circulación Cerebrovascular/efectos de los fármacos , Superóxido Dismutasa/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Encéfalo/irrigación sanguínea , Lesiones Encefálicas/diagnóstico por imagen , Dióxido de Carbono/sangre , Flujometría por Láser-Doppler , Masculino , Presión Parcial , Percusión , Ratas , Ratas Sprague-Dawley , UltrasonografíaRESUMEN
The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.
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Retículo Endoplásmico/ultraestructura , Proteínas de Choque Térmico , Canales Iónicos/ultraestructura , Proteínas de la Membrana/ultraestructura , Proteínas de Transporte de Membrana , Proteolípidos/ultraestructura , Proteínas de Saccharomyces cerevisiae , Animales , Transporte Biológico , Compartimento Celular , Detergentes , Perros , Técnica de Fractura por Congelación , Proteínas Fúngicas/metabolismo , Aumento de la Imagen , Activación del Canal Iónico , Sustancias Macromoleculares , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Peso Molecular , Movimiento (Física) , Coloración Negativa , Tamaño de la Partícula , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Ribosomas/metabolismo , Canales de Translocación SEC , LevadurasRESUMEN
The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT), media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.
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Fosfatidilcolina-Esterol O-Aciltransferasa/química , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Amidohidrolasas/metabolismo , Animales , Baculoviridae/genética , Células CHO , Carcinoma Hepatocelular , Colesterol/metabolismo , Cricetinae , Glicosilación , Humanos , Cinética , Neuraminidasa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/aislamiento & purificación , Fosfolipasas A/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transfección , Células Tumorales Cultivadas , Tunicamicina/farmacologíaRESUMEN
The tegument of the human parasite Schistosoma mansoni is critical for parasite survival within the mammalian host. The role of protein kinase C (PKC), a major effector molecule in the phosphoinositide pathway, in maintaining the structural organization of this syncytial layer was examined in adult worms. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB), phorbol esters that activate PKC, induced formation of surface vesicles as determined by light and scanning electron microscopy. Similar results were seen with sn-2-dioctanoyl-glycerol, a synthetic analogue of diacylglycerol. No effect was seen in parasites incubated with 4-alpha-phorbol ester or alpha isomers of PMA or PDB, compounds that do not activate PKC. Vesicle formation was reversible in parasites treated with sn-2-dioctanoyl-glycerol but not with phorbol esters. The tegument of male worms was more sensitive to the effect of phorbol esters than females. Transmission electron microscopy revealed vacuolization of the tegument. These data suggest that signal transduction pathways may have a critical role in the maintenance of the structural integrity of the tegument of parasitic helminths.
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Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/metabolismo , Schistosoma mansoni/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Biomphalaria/parasitología , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Schistosoma mansoni/enzimología , Schistosoma mansoni/ultraestructuraRESUMEN
The therapeutic process is a mix of technique and insight, and effective clinicians are able to blend theory with practice. This is especially true in the area of DAS. Children with the symptom complex of apraxia are a challenge to any therapy program. Clinicians should choose combinations of therapy protocols that best serve the child. MIT is one technique that has been shown to be successful.
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Apraxias/terapia , Logopedia , Niño , Preescolar , Humanos , MasculinoRESUMEN
By direct immunolabeling we have mapped the distribution of photosystem II (PS II) and cytochrome b6/f on the surfaces of photosynthetic membranes isolated from spinach. Photosynthetic membranes were attached to a support and gently disrupted to expose the occluded outer stacked surface, prior to labeling. Polyclonal antibodies against PS II intensely labeled the outer stacked surfaces while the outer nonstacked surface had minimal labeling. This confirms previous fractionation and immunolocalization studies which demonstrated that PS II is largely restricted to the stacked regions of the membrane. Inside-out membranes were also heavily labeled with PS II antibodies. Antibodies against cytochrome f were evenly distributed between the stacked and nonstacked outer surfaces and were found clumped together on the membrane outer surface. Previous fractionation and immunolocalization studies have indicated that cytochrome b6/f is located in both the stacked and nonstacked regions, but this is the first report to provide direct evidence that the complex may be clustered in the membrane. The clustering of antibodies to cytochrome b6/f supports the idea that this electron transport component exists as a multimeric complex within the membranes and that such complexes are found in both stacked and nonstacked regions of the photosynthetic membrane. No evidence was seen of any special differentiation of the marginal regions of the membrane, which link stacked and nonstacked regions.
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Citocromos/análisis , Membranas Intracelulares/ultraestructura , Orgánulos/ultraestructura , Proteínas del Complejo del Centro de Reacción Fotosintética/análisis , Plantas/ultraestructura , Citocromos f , Immunoblotting , Microscopía Inmunoelectrónica , Complejo de Proteína del Fotosistema IIRESUMEN
Titration calorimetry has been evaluated as a method for obtaining binding constants and thermodynamic parameters for the cytosolic fatty acid- and lipid-binding proteins. An important feature of this method was its ability to accurately determine binding constants in a non-perturbing manner. The equilibrium was not perturbed, since there was no requirement to separate bound and free ligand in order to obtain binding parameters. Also, the structure of the lipid-protein complex was not perturbed, since native ligands were used rather than non-native analogues. As illustrated for liver fatty acid-binding protein, the method distinguished affinity classes whose dissociation constants differed by an order of magnitude or less. It also distinguished endothermic from exothermic binding reactions, as illustrated for the binding of two closely related bile salts to ileal lipid-binding protein. The main limitations of the method were its relatively low sensitivity and the difficulty working with highly insoluble ligands, such as cholesterol or saturated long-chain fatty acids. However, the signal-to-noise ratio was improved by manipulating the buffer conditions, as illustrated for oleate binding to rat intestinal fatty acid binding protein. Binding parameters are reported for oleate interactions with several wild-type and mutant lipid-binding proteins from intestine. Where possible, the binding parameters obtained from calorimetry were compared with results obtained from fluorescence and Lipidex binding assays of comparable systems.
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Calorimetría/métodos , Proteínas Portadoras/metabolismo , Metabolismo de los Lípidos , Animales , Intestinos/química , Ratas , TermodinámicaRESUMEN
Cellular retinol-binding protein II (CRBP-II) and intestinal fatty acid-binding protein (I-FABP) are both expressed in small intestinal enterocytes and exhibit 31% sequence identity. I-FABP binds a single molecule of long-chain fatty acid and forms an ion-pair electrostatic interaction between the cationic side chain of arginine-106 and the anionic fatty acid carboxyl group. In contrast, CRBP-II binds all-trans-retinol or -retinal and contains a glutamine residue in the corresponding position, residue 109. We have characterized and compared the interactions of fatty acids and retinoids with I-FABP, CRBP-II, and two reciprocal mutant proteins. The mutants were designated CRBP-II(Q109R), where glutamine-109 was replaced by arginine, and I-FABP(R106Q), where arginine-106 was replaced by glutamine. As monitored by titration calorimetry and carbon-13 NMR spectroscopy, the fatty acid-binding properties of CRBP-II(Q109R) were found to be essentially identical to those of wild-type I-FABP. Both proteins bound 1 molecule of fatty acid with identical affinities (Kd = 0.2 microM). The enthalpic contribution to the total free energy of binding was large for both proteins: 66% and 87%, respectively. In addition, the carboxyl groups of fatty acids bound to both proteins were solvent-inaccessible. There was little or no change in the ionization state of the bound fatty acid over a wide pH range, as monitored by the chemical shift of the fatty acid carboxyl 13C resonance. Furthermore, the binding of fatty acid to both proteins was accompanied by a selective perturbation of the guanidino 13C resonance of a single arginine residue.(ABSTRACT TRUNCATED AT 250 WORDS)