RESUMEN
BACKGROUND: Complete following existing guidelines for management of acute coronary syndrome (ACS) is known to be associated with better outcomes. Partly this is explained by lesser adherence to recommendations in high risk patients. Aim of our study was to assess relationship between degree of following current guidelines and in hospital outcomes independently from initial assessment of risk. METHODS: Each key recommendation from guidelines issued between 2008 and 2011 (13 for STE ACS, 12 for NSTE ACS) was given weight of 1. Sum of these units constituted index of guideline adherence (IGA). IGA was retrospectively calculated for 1656 patients included in Russian independent ACS registry RECORD-2 (7 hospitals, duration 04.2009 to 04.2011). The patients were divided into 2 groups according to quartiles of IGA distribution: 1) low adherence group (quartiles I-II); 2) high adherence group (quartiles III-IV). RESULTS: In low adherence compared with high adherence group there were significantly more patients more or equal 65 years (=0.0007), with chronic heart failure [CHF] (<0.0001), previous stroke (<0.0001), atrial fibrillation [AF] (=0.0002), Killip class more or equal II (=0.0065), high risk of death by GRACE score (=0.035). Inhospital mortality was 9.3 and 2.4% in low and high adherence group, respectively (p<0.0001). The following independent predictors of inhospital death were identified: IGA quartiles I-II (odds ratio [OR] 4.0; 95% confidence interval [CI] 2.3-7.1; <0.0001), high GRACE score (OR 3.3; 95% CI 1.8-6.0; <0.0001), admission systolic BP less or equal 100 mm Hg (OR 3.1; 95% CI 1.8-5.4; <0.0001), admission serum glucose more or equal 8 mmol/l (OR 2.9; 95% CI 1.8-4.7; <0.0001), age more or equal 65 years (OR 2.3; 95% CI 1.3-4.0; =0.005), ST elevation more or equal 1 mm on first ECG (OR 1.7; 95% CI 1.1-2.5; =0.013). From groups with low and high adherence to guidelines we selected pairs of patients (n=588) with similar (or close) age, type of ACS, GRACE score, Killip class, presence of other important risk factors (CHF, AF, previous stroke), and formed 2 equal subgroups without significant differences in important demographic, anamnestic, clinical and laboratory data. Hospital mortality was 7.8 and 2.7% in low and high adherence subgroup, respectively (p<0.0001). CONCLUSIONS: In RECORD-2 ACS registry low adherence to guidelines was more frequent among high risk patients and was independent predictor of inhospital death. Association between degree of guidelines adherence and outcomes persisted after equalizing groups by some factors of risk of mortality.
Asunto(s)
Síndrome Coronario Agudo , Técnicas de Diagnóstico Cardiovascular , Adhesión a Directriz , Revascularización Miocárdica/métodos , Evaluación de Procesos y Resultados en Atención de Salud , Síndrome Coronario Agudo/diagnóstico , Síndrome Coronario Agudo/mortalidad , Síndrome Coronario Agudo/terapia , Anciano , Manejo de la Enfermedad , Femenino , Adhesión a Directriz/normas , Adhesión a Directriz/estadística & datos numéricos , Mortalidad Hospitalaria , Hospitalización/estadística & datos numéricos , Humanos , Masculino , Guías de Práctica Clínica como Asunto , Sistema de Registros/estadística & datos numéricos , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Federación de Rusia/epidemiología , Índice de Severidad de la EnfermedadRESUMEN
We have developed a simple method for fast analysis of single nucleotide polymorphisms and identification of target clones from cloned complex PCR products. The method utilizes Kamchatka crab duplex-specific nuclease and universal fluorescent probe and is alternative to laborious screening procedures using radioactive probes, restriction analysis followed by gel electrophoresis or expensive sequencing. The method efficacy was demonstrated in several model experiments.
Asunto(s)
Braquiuros/enzimología , Análisis Mutacional de ADN/métodos , ADN/genética , Desoxirribonucleasas/química , Polimorfismo de Nucleótido Simple/genética , Animales , Proteínas de Ciclo Celular/análisis , Clonación Molecular , ADN/química , Humanos , Proteínas Nucleares/análisis , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/análisis , Receptores de Antígenos de Linfocitos T/químicaRESUMEN
High contents of non-coding RNA in total bacteria RNA complicates considerably transcriptome analysis using standard approaches like high-throughput sequencing, gene expression profiles, subtractive hybridization. We suggest a procedure of preparation of bacterial cDNA for transcriptomics that includes rRNA and tRNA depletion with preservation of relative abundance of coding sequences. The method is based on the second order hybridization kinetics and unique properties of Kanchatka crab duplex-specific nuclease. The method efficacy was demonstrated on a model experiments.
Asunto(s)
ADN Complementario/aislamiento & purificación , Perfilación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Genoma Bacteriano , Células Procariotas/química , ARN no Traducido/químicaRESUMEN
Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades. In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was found that phagocytosis activates both cascades. The activation of Erkl/2 is dependent, and the activation of p38 is not dependent, on the activity of NADPH oxidase. Thus, it can be stated that the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism similar to that operating in nonphagocytizing cells, indicating the universality of the function of NADPH oxidases in different cell types.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , NADPH Oxidasas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Activación Enzimática , Sistema de Señalización de MAP Quinasas , Ratones , Fagocitos/metabolismo , FagocitosisRESUMEN
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.
Asunto(s)
ADN Complementario/química , ADN de Cadena Simple/química , Biblioteca de Genes , Animales , Braquiuros , Endonucleasas/química , Humanos , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
A family of genes of the agamic race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
Asunto(s)
Proteínas del Helminto/genética , Lectinas Tipo C/genética , Planarias/genética , Regeneración/fisiología , Secuencia de Aminoácidos , Animales , Proteínas del Helminto/metabolismo , Inmunohistoquímica , Intrones , Lectinas Tipo C/metabolismo , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Planarias/metabolismo , Planarias/ultraestructura , Regeneración/genéticaRESUMEN
The previously described gene sip1 belongs to transcription factors of the zinc finger family. It has been ascertained recently that this gene is involved in TGF signaling cascade. Mutations in human gene sip1 cause Hirschprung syndrome. The expression of gene sip1 during embryonic mouse development was studied by in situ hybridization and immunostaining. Starting at E12.5, sip1 transcripts are present in a number of tissues: in the cortical plate, ventricular zone of the basal ganglion, thalamus, pons and midbrain, in specific nuclei of the brain stem and in the dorsal part of the spinal cord. In the developing cerebral cortex, sip1 expression is region-specific. In the brain of adult mice, sip1 expression is mostly detected in hippocampus, dentate gyrus, and white matter of the neocortex. Sip1 protein expression in the cerebral cortex is mostly confined to glutamatergic neurons.
Asunto(s)
Sistema Nervioso Central/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Animales , Secuencia de Bases , Sistema Nervioso Central/embriología , Clonación Molecular , Cartilla de ADN , ADN Complementario , Hibridación in Situ , RatonesRESUMEN
The gene of a new red fluorescent protein zoan2RFP from a coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the Aequorea victoria jellyfish, was cloned. At early maturation stages, zoan2RFP exhibits a green fluorescence, which then turns into the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.
Asunto(s)
Antozoos/genética , Colorantes Fluorescentes/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Escherichia coli/genética , Fluorescencia , Proteínas Fluorescentes Verdes , Cinética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Mutación , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/química , Homología de Secuencia de Aminoácido , Espectrometría de FluorescenciaRESUMEN
A combination of suppression subtraction hybridization (SSH) and a new technique of mirror orientation selection (MOS) was used to compare the total DNA for two, sexual (SR) and asexual (AR), races of freshwater planarian Giradia tigrina. Several race-specific DNA fragments were found. A new element termed planarian extrachromosomal virus-like element (PEVE) was revealed in AR. The PEVE genome contains two unique regions, Ul and Us, which are flanked by inverted repeats. Two variants observed for the PEVE genome differ in combination of single- and double-stranded regions corresponding to Ul and Us. The PEVE genome codes for two helicases, one homologous to the circovirus replication initiation protein (Rep) and one corresponding to the helicase domain of papillomavirus E1. PEVE is nonuniformly distributed though the planarian body and is possibly replicated only in certain parenchymal cells.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Unión al ADN , Herencia Extracromosómica/genética , Planarias/genética , Secuencia de Aminoácidos , Animales , Cromosomas/genética , Circovirus/genética , Clonación Molecular , ADN Helicasas/genética , ADN de Cadena Simple , Agua Dulce , Orden Génico , Variación Genética , Hibridación in Situ/métodos , Datos de Secuencia Molecular , Papillomaviridae/genética , Planarias/fisiología , Secuencias Repetitivas de Ácidos Nucleicos , Reproducción/genética , Homología de Secuencia de Aminoácido , Transactivadores/genética , Virus/genéticaRESUMEN
The primary structure of the crusta gene encoding alpha-latrocrustoxin (alpha-LCT), a high molecular mass neurotoxin specific to crustaceans, was determined in the black widow spider Latrodectus mactans tredicimguttatus genome. The total length of the sequenced DNA was 4693 bp. The structural part of the black widow spider chromosome gene encoding alpha-LCT does not contain introns. The sequenced DNA contains a single extended open reading frame (4185 bp) and encodes a protein precursor of alpha-LCT, comprising 1395 aa. We assume the Met residue at position -10 relative to the N-terminal residue of Glu1 of the mature toxin to be the first one in the protein precursor. The calculated molecular mass of the precursor (156147 Da) exceeds that of the mature toxin by approximately 30 kDa. These data are in agreement with the notion that over the course of maturation the protein precursor undergoes double processing--cleavage of a decapeptide from the N-terminal part and of a approximately 200-aa fragment from the C-terminal part. alpha-LCT displayed a number of imperfect ankyrin-like repeats and areas of structural homology with earlier studied latrotoxins; the highest homology degree (62%) was revealed with alpha-latroinsectotoxin (alpha-LIT).
Asunto(s)
Araña Viuda Negra/genética , Venenos de Araña/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Genes de Insecto , Datos de Secuencia Molecular , Neurotoxinas/genética , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
The selective suppression of the polymerase chain reaction and methods based upon it (construction of cDNA libraries from low amounts of biological material, subtractive hybridization and differential display of mRNA, fast cloning of full-size cDNA, chromosome walking, cloning in vitro, and others) are reviewed. These methods display a high effectiveness and, taken together, enable intricate DNA analyses to be performed--from the search for nontrivial sequences to the total sequencing of the corresponding genes.
Asunto(s)
ADN Complementario/genética , Regulación de la Expresión Génica , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Paseo de Cromosoma , Cartilla de ADN , Biblioteca de Genes , Regiones Promotoras Genéticas , Supresión GenéticaRESUMEN
A new method for finding differentially expressed genes, termed ordered differential display of mRNAs (ODD), was used in the search for region-specific molecular markers of freshwater planarian Dugesia tigrina. In this method, the effect of selective suppression of a polymerase chain reaction (PCR) is used for the differential amplification of a pool of 3'-terminal cDNA fragments generated by digestion of cDNAs with a restriction endonuclease. In the resulting amplified cDNAs, every mRNA is represented by a cDNA fragment whose length is determined by the position of the restriction site nearest to the 3'-terminus. Subsequent PCR with primers 3'-extended by two random nucleotides allowed the amplification of 1/192 part of all cDNA molecules present in the sample. The comparison of the generated pools of cDNA molecules separated by PAGE leads to the identification of differentially expressed sequences. The systematic study of the total mRNA pool is achieved by the successive use of all possible combinations of extended primers. Some sequences preferentially expressed along the anterior-posterior axis of planarian were identified using ODD.
Asunto(s)
Clonación Molecular/métodos , Marcadores Genéticos/genética , Planarias/genética , Animales , Secuencia de Bases , Cartilla de ADN , ADN Complementario , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A method of construction of amplified cDNA libraries was developed on the basis of selective inhibition of cDNA amplification and modified for the studied models for analysis of expression of the genes containing LeR-1 and VeR-1 sequences. Time-related changes in expression of these genes were studied during regeneration of the adult lens and during embryogenesis of newts. The pattern of expression of the LeR-1 and VeR-1 genes proved to be not tissue-specific. A fragment adjoining 5'-area of the LeR-1 gene was obtained using a new approach: rapid amplification of cDNA ends by polymerase chain reaction (5'-RACE-PCR). Analysis of the LeR-1 clone primary structure using Gene Bank did not show essential homology with the known nucleotide sequences. Sequence LeR-1 is characterized by evolutionary conservation of genomic DNA in Pleurodeles waltlii, Xenopus laevis, and Rana temporaria.
Asunto(s)
Desarrollo Embrionario y Fetal/genética , Regulación del Desarrollo de la Expresión Génica/genética , Cristalino/fisiología , Regeneración/genética , Salamandridae/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Amplificación de Genes/genética , Datos de Secuencia Molecular , Pleurodeles , Reacción en Cadena de la Polimerasa , ARN/genética , Rana temporaria , Salamandridae/embriología , Xenopus laevisRESUMEN
We identified two new genes (scarf and collar) of planarians using subtracting hybridization. mR-NAs of these genes are distributed in different ways in regeneration blastemas of different polarity. Zone-specific expression of these genes in non-regenerating planarians has been demonstrated. The level of expression of the identified genes in the head regeneration buds of the first-third days of regeneration was lower than that in the corresponding intact tissues and tail regeneration buds.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Planarias/genética , Regeneración/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Polaridad Celular/genética , ADN Complementario/genética , ADN de Helmintos/genética , Biblioteca de Genes , Marcadores Genéticos/genética , Hibridación Genética/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
An improved version of the in vitro DNA cloning method described earlier is proposed. The method allows amplification by polymerase chain reaction (PCR) of individual DNA molecules of an unknown primary structure followed by sequencing. The modifications described here provide for in vitro cloning by means of a 40-45-cycle PCR (the original protocol required two consecutive amplifications). In addition, the in vitro cloning is suggested to be carried out in special 96-well plates in the presence of ethidium bromide; upon UV irradiation, the wells containing amplified DNA fluoresce to make the analysis of all 96 wells unnecessary. The improved protocol makes the preparation of individual in vitro clones more straightforward and less expensive.
Asunto(s)
Clonación Molecular/métodos , ADN Complementario/química , Reacción en Cadena de la Polimerasa , Secuencia de Bases , ADN Complementario/genética , Electroforesis en Gel de Agar , Etidio/química , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Rayos UltravioletaRESUMEN
A novel, efficient and simple technique that combines subtractive hybridization with kinetic enrichment is proposed for obtaining enriched cDNA. The method is based on the use of a set of special primers that allow for the selective amplification by PCR only of differentially distributed sequences. Using the proposed technique, cDNA of a new gene XEp-1, specifically expressed in the presumptive epidermis of Xenopus laevis, was cloned, starting with the stage of midgastrula.
Asunto(s)
ADN Complementario/genética , Expresión Génica , Genes Homeobox , Hibridación de Ácido Nucleico/métodos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Xenopus laevisRESUMEN
A new efficient method for obtaining cDNA libraries with equal representation of all cDNA types (equalized libraries) in a single round of equalization was developed. The method is based on differences in the renaturation kinetics of double-stranded cDNAs of different genes and allow the selection of the equalized single-stranded fraction resulted from the incomplete reassociation of the total cDNA without laborious and inefficient physical separation. The equalized single-stranded fractions are selectively amplified by polymerase chain reaction (PCR). The amplification of other DNA molecules is inhibited due to PCR suppression, i.e. the suppression of amplification of the DNA molecules flanked with long interval terminal repeats in PCR with a primer corresponding to the external moiety of the repeat. The efficiency of the developed method was estimated in obtaining an equalized cDNA library based on mRNA from the activated human T lymphocyte Jurkat cell line.
Asunto(s)
ADN Complementario/genética , Biblioteca de Genes , Reacción en Cadena de la Polimerasa , Supresión Genética , Secuencia de Bases , ADN de Cadena Simple/genética , Humanos , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/genéticaRESUMEN
Using subtractive hybridization, a cDNA library containing over 50% of clones specific for a highly metastatic cell line was obtained from two hamster embryo fibroblast lines with different metastatic potentials. Most of the clones (83%) contained new sequences. One clone contained the ha-SDGF gene cDNA homologous to SDGF cDNA from rodents. The level of ha-SDGF mRNA expression was considerably higher in the highly metastatic cell line.
Asunto(s)
Glicoproteínas , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Secuencia de Aminoácidos , Anfirregulina , Animales , Línea Celular Transformada , Clonación Molecular , Cricetinae , ADN Complementario , Familia de Proteínas EGF , Humanos , Mesocricetus , Datos de Secuencia Molecular , Metástasis de la Neoplasia/genética , Hibridación de Ácido Nucleico , Roedores , Homología de Secuencia de AminoácidoRESUMEN
An efficacious method of cloning the sequences common for two cDNAs was proposed. The method was used for constructing a library of expressed sequences evolutionarily conserved for human and hamster.
Asunto(s)
Evolución Biológica , Secuencia Conservada , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , ADN Complementario , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
During lens regeneration in Pleurodeles waltl, the dorsal iris zone is the cell source of the lens regeneration, while the ventral iris zone can serve as the cells' source of lens regeneration only under certain experimental conditions. The method of subtractive hybridization was used for the identification of genes responsible for the different proliferative potential of these zones. Differential screening of the enriched cDNA libraries, which were obtained as a result of subtractive hybridization of the cDNA samples of the ventral and dorsal iris zones 14 days after lens removal, revealed four clones specific to the dorsal iris and six clones specific to the ventral iris. Two of these, LeR-1 and VeR-1, were structurally characterized. Comparison of their primary structure with data from the Gene Bank showed no essential homology with the known sequences. Time-related changes in LeR-1 and VeR-1 expression were shown during lens regeneration. LeR-1 and VeR-1 expression was activated at the early stages of lens regeneration. The peaks of LeR-1 and VeR-1 expression were observed on the 14th day of lens regeneration in the dorsal and ventral iris zones, respectively. Furthermore, LeR-1 is activated during retina regeneration. The results of Southern hybridization suggest the presence of sequences complementary to LeR-1 in the genomes of Pleurodeles waltl and Rana temporaria. We propose that the activation of LeR-1 expression is related to the triggering of lens regeneration, while the activation of VeR-1 expression accompanies the inhibition of proliferative activity in the ventral iris zone.