Mejora de la Señal de Flujo Sanguíneo en Implantes Coronarios Mediante la Detección de Distorsiones Eventuales / Improving the Blood Flow Signal in Coronary Bypass by Detection of Eventual Distortions

Se presenta un método para el procesamiento de señales en aplicaciones médicas que utilizan técnicas de diagnóstico basadas en ultrasonido Doppler. El método está orientado a obtener una mejor representación de la señal de flujo sanguíneo, a partir de la identificación y exclusión de los ciclos, de dicha señal, que se encuentran afectados por distorsiones eventuales. Esto permite, de manera robusta y confiable, estimar parámetros y extraer información clínicamente útil con el objetivo de formular diagnósticos precisos sobre la funcionalidad del objeto examinado. Se muestran los resultados de la aplicación del método sobre señales reales de flujo sanguíneo obtenidas durante procedimientos de revascularización coronaria y se demuestra que la estimación de los índices clínicos de interés mejora considerablemente cuando los ciclos detectados como afectados por ruido eventual son excluidos del análisis.

This paper aims to implement a method for signals processing in biomedical applications based on Doppler ultrasound techniques. The method is aimed to obtain a better representation of the blood flow signal, and it is based on the detection and exclusion of signal cycles detected as affected by eventual noise. This allows, in robust and reliable way, to estimate parameters and extract useful clinical information, in order to make accurate diagnoses on the functionality of the scanned object. The results of applying the method to two blood flow signals in coronary implants are presented, and it is observed that the estimation of the clinical indices improve when affected cycles are excluded of the signal analysis.

In-vivo stimulation of macaque natural killer T cells with α-galactosylceramide.

Natural killer T cells are a potent mediator of anti-viral immunity in mice, but little is known about the effects of manipulating NKT cells in non-human primates. We evaluated the delivery of the NKT cell ligand, α-galactosylceramide (α-GalCer), in 27 macaques by studying the effects of different dosing (1-100 µg), and delivery modes [directly intravenously (i.v.) or pulsed onto blood or peripheral blood mononuclear cells]. We found that peripheral NKT cells were depleted transiently from the periphery following α-GalCer administration across all delivery modes, particularly in doses of ≥10 µg. Furthermore, NKT cell numbers frequently remained depressed at i.v. α-GalCer doses of >10 µg. Levels of cytokine expression were also not enhanced after α-GalCer delivery to macaques. To evaluate the effects of α-GalCer administration on anti-viral immunity, we administered α-GalCer either together with live attenuated influenza virus infection or prior to simian immunodeficiency virus (SIV) infection of two macaques. There was no clear enhancement of influenza-specific T or B cell immunity following α-GalCer delivery. Further, there was no modulation of pathogenic SIVmac251 infection following α-GalCer delivery to a further two macaques in a pilot study. Accordingly, although macaque peripheral NKT cells are modulated by α-GalCer in vivo, at least for the dosing regimens tested in this study, this does not appear to have a significant impact on anti-viral immunity in macaque models.

Metal-metal bonded diruthenium(II, III) assemblies with the polycyano anionic linkers N(CN)2-, C(CN)3-, and 1,4-dicyanamido-2,5-dimethylbenzene (DM-dicyd2-): syntheses, structures, and magnetic properties.

The new metal-metal bonded diruthenium(II,III) compounds [Ru2(O2CCH3)4(mu-L)]infinity (L = N(CN)2-, 1; C(CN)3-, 2) and [[Ru2(O2CCH3)2(mhp)2]2(mu-DM-Dicyd)] (3) (mhp = 2-oxy-6-methylpyridinate, DM-Dicyd = 1,4-dicyanamido-2,5-dimethylbenzene dianion) have been synthesized and fully characterized. Compounds 1 and 2 were synthesized by the reaction of [Ru2(O2CCH3)4(NCCH3)2](BF4) with NaN(CN)2 and KC(CN)3, respectively. The "dimer-of-dimers", 3, was synthesized by a 2:1 reaction of [Ru2(O2CCH3)2(mhp)2(MeOH)](BF4) with [As(Ph)4]2[DM-Dicyd]. Compound 1 crystallizes in the monoclinic space group C2/m with a = 10.174(2) A, b = 13.016(3) A, c = 7.0750(14) A, beta = 101.83(3) degrees, and Z = 2. Compound 2 crystallizes in the orthorhombic space group Fdd2 with a = 29.679(6) A, b = 31.409(6) A, c = 7.3660(15) A, V = 6866(2) A3, and Z = 16. In compound 1, dicyanamide anions (N(CN)2-) bridge the [Ru2(O2CCH3)4]+ units in an end-to-end bridging mode, thereby forming an alternating one-dimensional chain. In compound 2, two cyano groups of tricyanomethanide anion (C(CN)3-) are coordinated to independent [Ru2(O2CCH3)4]+ units to give a chain similar to that found in 1. The Ru-Ru bond distances in 1 and 2 are 2.2788(14) and 2.2756(5) A, respectively, which are typical values for Ru2(O2CR)4Cl and [Ru2(O2CR)4]+ compounds. The Ru-N distances are 2.257(8) A in 1 and 2.259(4) and 2.283(4) A in 2. The temperature dependence of the magnetic susceptibilities of compounds 1-3 reveals a weak antiferromagnetic interaction between Ru2 units (S = 3/2) through each polycyano anionic linker: g = 2.16, zJ = -0.33 cm(-1), D = 63.3 cm(-1) for 1; g = 2.15, zJ = -0.22 cm(-1), D = 58.0 cm(-1) for 2; and g = 2.10, zJ = -0.90 cm(-1), D = 75.0 cm(-1) for 3.

Estudio postmortem de variables morfo-métricas de los lóbulos frontal y temporal en encéfalos de pacientes esquizofrénicos / Postmortem study of morphometric parameters of frontal and temporal lobes on schizophrenic patiens

La esquizofrenia era considerada antiguamente una psicosis de tipo funcional; sin embargo en la actualidad se conoce que los encéfalos de estos pacientes presentan cambios morfológicos, tanto macroscópicos como microscópicos, entre los que destacan la disminución del peso encefálico, la dilatación ventricular y la atrofia cortical. Con el objetivo de caracterizar un poco más este proceso atrófico se realizó un estudio morfométrico postmortem de 36 encé-falos de pacientes esquizofrénicos y 30 encéfalos de sujetos controles, a los cuales se les realizaron dos cortes frontales para estudiar algunas variables de los lóbulos frontal y temporal. No se encontraron diferencias entre los sujetos esquizofrénicos y controles en cuanto a los pesos encefálico, cerebral y de cada hemisferio. Se observó una disminución del área de los lóbulos frontales y del lóbulo temporal derecho en los cerebros de personas esquizofrénicas; sin embar-go, en este grupo no se halló dilatación ventricular ni signos de atrofia cortical ni central. Ninguno de los signos de asimetría cerebral encontrados en el grupo control apareció en el grupo esquizofrénico (AU)

Cyclosporine-induced retinal toxic blindness.

We report the case of a BMT recipient who developed blindness 22 months after BMT. Microvascular retinopathy, cortical blindness and other ocular pathologies were excluded with appropriate tests. Electrophysiological studies showed retinal damage without excluding an optic nerve lesion. The patient, who had several risk factors for neurologic-induced cyclosporine toxicity, improved with cyclosporine withdrawal. Our findings stress the need of electrophysiological tests to exclude neuroretinal damage in patients receiving cyclosporine after BMT.

AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and non-catalytic, beta and gamma (38 kDa) subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and we find that rat liver AMPK-alpha2 isoform catalytic subunit is associated with beta1 and gamma1 and not with beta2 or gamma2 subunit isoforms. The beta1 and gamma1 isoforms are also subunits of the alpha1 isoform. The sequence of cloned human AMPK-beta1 is 95% identical in amino acid sequence with rat beta1. Human chromosomal localizations were determined for AMPK-alpha1 (5p11-p14), AMPK-beta1 (12q24.1-24.3) and AMPK-gamma1 (12q12-q14), respectively.

Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2.

SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.

Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits.

The mammalian 5'-AMP-activated protein kinase is a heterotrimer consisting of an alpha catalytic subunit and beta and gamma noncatalytic subunits, each of which is represented in a larger isoprotein family, related to the SNF1 kinase and its interacting proteins in yeast. In this study, we have used mammalian cell transfection to compare the activities of the two alpha subunit isoforms, alpha-1 and alpha-2, and to study the influence of the noncatalytic subunits on enzyme subunit association and activity. Expression of epitope-tagged protein subunits in COS7 cells indicates detectable but low level kinase activity for each of the two catalytic alpha subunits. Co-expression of alpha subunits with the beta or gamma subunits modestly increases kinase activity accompanied by the formation of alpha/beta or alpha/gamma heterodimers. Co-expression of all three subunits, however, is accompanied by a 50-110-fold increase in kinase activity with the formation of a heterotrimeric complex. In addition to binding of each noncatalytic subunit to the alpha subunit, the beta and gamma subunits bind to each other, likely resulting in a more stable heterotrimeric complex. The increase in kinase activity associated with expression of this heterotrimer is due both to an increase in enzyme-specific activity (units/enzyme mass) and to an apparent enhanced alpha subunit expression. Co-expression of a catalytically defective alpha subunit or the beta/gamma-binding COOH-terminal domain of the alpha subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic beta and gamma subunits of 5'-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf1p and Snf4p seem to be required for maximum kinase activity.

Non-catalytic beta- and gamma-subunit isoforms of the 5'-AMP-activated protein kinase.

The mammalian 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of alpha-, beta-, and gamma-subunits. The alpha-subunit is the catalytic subunit and is related to the yeast Snf1p kinase. In this study, we report the cloning of full-length cDNAs for the non-catalytic beta- and gamma-subunits. The rat liver AMPK beta-subunit clone predicts a protein of 30,464 Da, which is related to the Sip1p, Sip2p, and Gal83p subfamily of yeast proteins that interact with Snf1p and are involved in glucose regulation of gene expression. The AMPK beta-subunit, when expressed in bacteria and in mammalian cells, migrates anomalously on SDS gels at an apparent molecular mass of 40 kDa. Rat and human liver AMPK gamma-subunit clones predict a protein of 37,577 Da (AMPK-gamma1), which is related to the yeast Snf4p protein that copurifies with Snf1p and to a larger family of other human AMPK gamma-isoforms. The mRNAs for both AMPK- beta and AMPK-gamma1 are widely expressed in rat tissues, consistent with a broad role for AMPK in cellular regulation. These data reveal a mammalian multisubunit protein kinase strikingly similar to the multisubunit glucose-sensing Snf1 kinase complex. The identification of isoform families for the AMPK subunits indicates the potential diversity of the roles of this highly conserved signaling system in nutrient regulation and utilization in mammalian cells.

Mammalian AMP-activated protein kinase subfamily.

The mammalian 5'-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (alpha 1) is distinct from the previously cloned kinase subunit (alpha 2). The alpha 1 (548 residues) and alpha 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase alpha 1 mRNA distribution and the alpha 1 immunoreactivity rather than alpha 2, with substantial amounts in kidney, liver, lung, heart, and brain. Both alpha 1 and alpha 2 isoforms are stimulated by AMP and contain noncatalytic beta and gamma subunits. The liver alpha 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the alpha 2 immunoreactivity parallels the alpha 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the beta and gamma subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.

Mammalian homologues of the Drosophila Son of sevenless gene map to murine chromosomes 17 and 12 and to human chromosomes 2 and 14, respectively.

Activating mutations in the ras genes are commonly found in a wide range of human tumors. We recently cloned two mammalian genes, Son of sevenless 1 (mSos1) and Son of sevenless 2 (mSos2), whose protein products appear to be important positive regulators of ras proteins. Given the proposed role of Sos proteins in ras regulation, and the frequent occurrence of activated ras alleles in tumor cells, we were interested in determining whether the Sos genes may also be activated inappropriately by DNA rearrangement in tumor cells. To investigate this possibility, we have determined the chromosomal locations of both the mouse and the human Sos1 and Sos2 genes, using a combination of genetic linkage analysis and in situ hybridization to chromosomal spreads. We find that the murine Sos1 and Sos2 genes map to chromosomes 17E and 12C3.3-D and their human counterparts to chromosomes 2p21-2p2 and 14q21, respectively. Neither the human nor the mouse Sos loci map close to known mutations or to regions showing consistent karyotypic abnormalities in tumor cells.

High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli.

Synthesis in E. coli of native coat protein of Johnsongrass mosaic virus, and hybrid protein molecules containing foreign antigens, resulted in the intracellular formation of potyvirus-like particles (PVLPs). The foreign antigens used were an octapeptide epitope from Plasmodium falciparum and a decapeptide hormone (luteinizing hormone releasing hormone) at the N- or at both N- and C-terminal regions of the coat protein molecule, and a full length protein antigen (Sj26-glutathione S-transferase of 26 kD from Schistosoma japonicum) replacing the N-terminal 62 amino acids of the coat protein. Electron microscopy of ultrathin sections of E. coli revealed that PVLPs resulting from coat protein molecules containing peptide fusions appeared in vast arrays of parallel strands within the cytoplasm sometimes extending the length of the cell and at times the cells were strung together, with "threads" of PVLPs appearing to connect individual bacterial cells. PVLPs resulting from the fusion of the 26 kD antigen Sj26 to coat protein were shorter and wider. The physical form of the high molecular weight PVLPs enabled purification by simple size exclusion column chromatography. The Sj26-PVLPs administered to mice without adjuvant elicited antibody responses comparable to monomeric Sj26 administered with Freund's Complete Adjuvant.

Immunologic and biologic characteristics of human decidual prolactin.

An increasing and consistent production of prolactin by decidual tissue (dhPRL) has been shown during a 24-hour incubation in a chemically defined buffer medium. No difference in dhPRL release was observed in tissue obtained from first- and third-trimester pregnancy. Estradiol had neither a stimulatory nor an inhibitory influence on dhPRL release, although release of this polypeptide by decidual tissue incubated with cycloheximide was blocked. The precise mechanism controlling the production and release of dhPRL is not known. Addition of theophylline, dibutyryl cyclic adenosine monophosphate, or guanosine triphosphate had no effect on dhPRL release during a 4-hour incubation. In contrast, these substances provoked a statistically significant increase in PRL release from rat hemipituitaries coincubated in vitro. Displacement curves for dhPRL against a human PRL standard suggest molecular homogeneity between both hormones. The biologic activity of dhPRL was next confirmed by means of a new bioassay system with a lower level of sensitivity of 20 pg/ml. In addition, dissociation of the biologic and immunologic activity of dhPRL has been completed and suggests that at least three isohormones are produced by human decidual tissue. The dhPRL production rate per gram of decidual tissue wet weight is far below that reported for the pituitary homologue. These studies not only provide confirmatory evidence of the immunologic and biologic activity of dhPRL but also intensify speculation as to its role in human gestation, including that related to osmoregulation across human amnion.