RESUMEN
Routine varicella vaccination with one dose for children of 11 to 14 months was recommended in Germany in 2004 to reduce disease incidence and severe complications. A country-wide varicella sentinel surveillance system was initiated in 2005 to detect trends of disease frequency and vaccine uptake and to evaluate the vaccination programme. A convenient sample of about 1,000 paediatricians and general practitioners was recruited to report on a monthly basis on varicella cases by age groups seen in their practice, and on varicella vaccine doses administered. Sentinel data from April 2005 to March 2009 show a reduction of 55% of varicella cases in all ages; 63% in the age group 0-4 years and 38% in 5-9-year-olds. The number of vaccine doses per reporting unit in all regions and physician groups increased during the same period. The number of reported cases as well as administered vaccines differed between physician groups and regions with different reimbursement policies. Where reimbursement was settled early and vaccine doses were increasing varicella cases started to decrease early as well. Besides reimbursement policies the availability and vaccination schedules influenced vaccine uptake. Sentinel surveillance provided valid data on trends for varicella associated morbidity, vaccine uptake and the age distribution of cases. The results confirm that following the introduction of routine varicella vaccination, varicella morbidity started to decline in Germany.
Asunto(s)
Vacuna contra la Varicela/uso terapéutico , Varicela/epidemiología , Varicela/prevención & control , Vacunación/tendencias , Adolescente , Niño , Preescolar , Alemania/epidemiología , Humanos , Lactante , Vacunación/métodos , Adulto JovenRESUMEN
It is evident that the complete elimination of measles, mumps and varicella has not yet been accomplished, as various outbreaks have shown. In the recent past the number of infections of teenagers and adults with these so called 'children's diseases' have been increasing. The course of the infections in these cases are often severe. To improve the current situation it will be necessary to: strictly undertake (re-)vaccination of all persons who may not be protected; extensive immunization for occupational indications (including apprentices, trainees and students); broad postexposure vaccinations; outbreak response immunizations of persons having no proof of proper vaccination (twice) or immunity. Teenagers and adults, in addition to children should obtain protection by being immunized against measles, mumps, rubella and chickenpox (varicella).
Asunto(s)
Vacuna contra la Varicela/uso terapéutico , Vacuna Antisarampión/uso terapéutico , Vacuna contra la Parotiditis/uso terapéutico , Adolescente , Adulto , Varicela/epidemiología , Varicela/inmunología , Vacuna contra la Varicela/administración & dosificación , Niño , Alemania/epidemiología , Humanos , Sarampión/epidemiología , Sarampión/inmunología , Vacuna Antisarampión/administración & dosificación , Paperas/epidemiología , Paperas/inmunología , Vacuna contra la Parotiditis/administración & dosificaciónRESUMEN
Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Cadena A de alfa-Cristalina/análisis , Cadena A de alfa-Cristalina/química , Animales , Cristalino/química , Ratones , Estructura Terciaria de Proteína , Cadena A de alfa-Cristalina/aislamiento & purificaciónRESUMEN
Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mycobacterium tuberculosis/química , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/fisiología , Proteómica/métodos , Transducción de Señal , Especificidad por SustratoRESUMEN
Anemia of chronic disease (ACD) is a frequent complication of chronic inflammation in rheumatoid arthritis (RA). Recombinant human erythropoietin (rHuEpo) has been shown to be effective in correcting ACD, although with a variable rate of nonresponders. The first aim of this trial was to improve the response to rHuEpo by parenteral iron supplementation in cases of iron-deficient erythropoiesis (IDE). An additional goal was the evaluation of the zinc protoporphyrin content of erythrocytes (ZnPP), the soluble transferrin receptor (sTrfR) serum concentration, and the hemoglobin (Hb) content of reticulocytes (CHr) in stimulated erythropoiesis as diagnostic and prognostic parameters. Thirty RA patients with ACD were treated with subcutaneous 150 IU rHuEpo/kg body weight twice weekly. Intravenous iron supplementation (200 mg iron sucrose once weekly) was added in cases of IDE (n=23), which was defined by the presence of two of three criteria: saturation of transferrin (TrfS) < or =15%, hypochromic erythrocytes (HypoE) > or =10%, and a serum ferritin (Fn) concentration < or =50 microg/l. All 28 completers met the treatment goal, with an increase of the median Hb concentration from 10.3 g/dl to 13.3 g/dl. Epo treatment and iron supplementation was safe and well tolerated in all patients. Monitoring of Fn, TrfS, and HypoE every other week allowed a successful correction of anemia. Retrospective analysis of the evaluable parameters (CHr, sTrfR, and ZnPP) revealed no additional benefit for predicting or monitoring IDE in this setting, although the one or other may be advantageous in other therapeutic situations.
Asunto(s)
Anemia/tratamiento farmacológico , Eritropoyesis/efectos de los fármacos , Eritropoyetina/administración & dosificación , Adulto , Anciano , Anemia/etiología , Artritis Reumatoide/sangre , Artritis Reumatoide/complicaciones , Biomarcadores/sangre , Enfermedad Crónica , Femenino , Ferritinas/sangre , Hemoglobinas/análisis , Humanos , Hierro/administración & dosificación , Deficiencias de Hierro , Masculino , Persona de Mediana Edad , Protoporfirinas/sangre , Receptores de Transferrina/sangre , Proteínas Recombinantes , ReticulocitosRESUMEN
At the 54(th) Scientific Congress of the German Professional Association of Public Health Service Physicians and Dentists in Marburg on 6th May 2004 the working group on meningococci (Arbeitsgemeinschaft Meningokokken, AGMK) organised the international workshop "Public Health Management of invasive Meningococcal Disease". In recent years significant changes in the epidemiology of meningococcal disease took place in Europe: in some countries and regions the number of disease caused by meningococci serogroup C has increased significantly. In the Netherlands this increase led to the introduction of an immunisation programme with conjugated meningococcal vaccines targeting children aged 1 up to 18 years. In Switzerland a peak in the number of reported meningococcal group C cases could be observed in some regions. Therefore, a regional vaccination programme has been introduced. Nevertheless, compared with Germany, the indications for vaccination against meningococci in Switzerland are more extensive. In the workshop, Professor Ulrich Vogel and Dr. Ingrid Ehrhard presented the epidemiological situation in Germany and the recommended prophylaxis regimen against meningococci.
Asunto(s)
Control de Enfermedades Transmisibles , Notificación de Enfermedades/estadística & datos numéricos , Meningitis Meningocócica/prevención & control , Vacunas Meningococicas/administración & dosificación , Vigilancia de la Población , Adolescente , Niño , Preescolar , Estudios Transversales , Europa (Continente)/epidemiología , Femenino , Alemania/epidemiología , Humanos , Incidencia , Lactante , Masculino , Meningitis Meningocócica/epidemiología , Meningitis Meningocócica/inmunología , Neisseria meningitidis Serogrupo C/inmunologíaRESUMEN
Nowadays undisputed is the effectiveness of early and long-standing DMARD therapy in the presence of active rheumatoid arthritis on disease progression and avoidance of structural joint changes. "Early" is defined as immediate initiation of drug therapy after diagnosis of rheumatoid arthritis. "Long-term" refers to a mostly life-long therapy, which even in the case of remission should be continued for at least 1 year. Clinical and laboratory routine controls during DMARD therapy are absolutely necessary. "DMARDs" summarize disease-modifying antirheumatic drugs such as methotrexate, sulfasalazine, leflunomide, hydroxychloroquine, aurum but also the TNF-blockers infliximab and etanercept. In cases of disease remission with combination drug therapy, corticosteroids and NSAID should be discontinued in a timely manner ahead of DMARDs to ensure that the reduction of clinical symptoms is not steroid controlled. DMARD therapy should end at least 6 months prior to conception.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Antiinflamatorios/administración & dosificación , Antiinflamatorios/efectos adversos , Antiinflamatorios/uso terapéutico , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/uso terapéutico , Antirreumáticos/administración & dosificación , Antirreumáticos/uso terapéutico , Artritis Reumatoide/diagnóstico , Ensayos Clínicos Controlados como Asunto , Quimioterapia Combinada , Glucocorticoides/administración & dosificación , Glucocorticoides/efectos adversos , Glucocorticoides/uso terapéutico , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Metotrexato/administración & dosificación , Metotrexato/uso terapéutico , Estudios Retrospectivos , Factores de TiempoRESUMEN
Helicobacter pylori is a very common bacterial pathogen that causes gastric disease by inducing the infiltration of immune cells as an initial event. Virulent H. pylori strains express a type IV secretion system composed of several virulence (Vir) proteins encoded by the cag pathogenicity island (cag PAI). During infection of phagocytic cells (U937, Josk-M and J774A.1) we have detected a de novo tyrosine-phosphorylated protein (p35p-Tyr) with sizes of 30 kDa, 38 kDa or 40 kDa, depending on the H. pylori strain. p35p-Tyr occurrence required functional virB4, virB7, virB10, virB11, virD4 and cagA (cytotoxin-associated gene A) genes encoded by the cag PAI suggesting that p35p-Tyr is a bacterial protein of variable size. We have biochemically purified p35p-Tyr from infected U937 cells. Tryptic peptides of p35p-Tyr determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) identified the carboxy (C)-terminal part of the H. pylori CagA protein. Subsequent analysis by two-dimensional electrophoresis (2-DE) and immunoblotting using anti-CagA antibodies revealed the presence of three stable CagA protein species in phagocytes: (i) 130-140 kDa full-length CagA (p135CagA), (ii) a 100-105 kDa fragment (p100CagA) and (iii) a 30-40 kDa fragment (p35CagA). Unlike p135CagA, p35CagA and p100CagA were also detected in much lower amounts in H. pylori without host cell contact. Therefore, breakage or processing leads to the production of p35CagA and p100CagA, a process that is enhanced after translocation into host cells. MALDI-MS data and the isoelectric point determined by both 2-DE and sequence analysis suggested that p35CagA represents the C-terminal part of CagA and p100CagA corresponds to the remaining amino (N)-terminal fragment. The possible function of CagA in host signal transduction and development of gastric disease is discussed.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Helicobacter pylori/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Línea Celular , Electroforesis en Gel Bidimensional , Genes Bacterianos , Infecciones por Helicobacter/etiología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Immunoblotting , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/genética , Mapeo Peptídico , Fagocitos/metabolismo , Fagocitos/microbiología , Fosforilación , Procesamiento Proteico-Postraduccional , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tirosina/metabolismo , VirulenciaRESUMEN
A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.
Asunto(s)
Proteínas Bacterianas/análisis , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Mycobacterium bovis/química , Mycobacterium tuberculosis/química , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Eliminación de Gen , Genes Bacterianos , Genoma Bacteriano , Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidad , Mycobacterium tuberculosis/genética , Técnica de Sustracción , Virulencia/genéticaRESUMEN
Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.
Asunto(s)
Proteínas Bacterianas/análisis , Helicobacter pylori/química , Proteoma/análisis , Proteínas Bacterianas/química , Bases de Datos Factuales , Electroforesis en Gel Bidimensional , Helicobacter pylori/genética , Helicobacter pylori/crecimiento & desarrollo , Humanos , Concentración de Iones de Hidrógeno , Immunoblotting , Peso Molecular , Sistemas de Lectura Abierta , Proteoma/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Helicobacter pylori is one of the most common bacterial pathogens, infecting about 50% of the world population. The presence of a pathogenicity island (PAI) in H. pylori has been associated with gastric disease. We present evidence that the H. pylori protein encoded by the cytotoxin-associated gene A (cagA) is translocated and phosphorylated in infected epithelial cells. Two-dimensional gel electrophoresis (2-DE) of proteins isolated from infected AGS cells revealed H. pylori strain-specific and time-dependent tyrosine phosphorylation and dephosphorylation of several 125-135 kDa and 75-80 kDa proteins. Immunoblotting studies, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), cell fractionation and confocal microscopy demonstrated that one of the 125-135 kDa proteins represents the H. pylori CagA protein, which is translocated into the host cell membrane and the cytoplasm. Translocation of CagA was dependent on functional cagA gene and virulence (vir) genes of a type IV secretion apparatus composed of virB4, virB7, virB10, virB11 and virD4 encoded in the cag PAI of H. pylori. Our findings support the view that H. pylori actively translocates virulence determinants, including CagA, which could be involved in the development of a variety of gastric disease.
Asunto(s)
Adenocarcinoma/microbiología , Antígenos Bacterianos , Proteínas Bacterianas/metabolismo , Células Epiteliales/microbiología , Helicobacter pylori/patogenicidad , Neoplasias Gástricas/microbiología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Citoplasma/metabolismo , Electroforesis en Gel Bidimensional , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Humanos , Datos de Secuencia Molecular , Fosforilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Células Tumorales CultivadasRESUMEN
Data are presented from a novel microfocus X-ray generator installed with a choice of ellipsoidal specularly reflecting mirrors. Diffraction data from proteins show the useful flux from this low-power device to be approaching equivalence with that from many far more powerful generators. Intensity measurements show that for small crystals the brilliance is now restricted by the performance of the mirror, which appears to be limited by imperfections in the figure of its surface rather than by a low reflectivity. Suitable choices of ellipsoidal mirror enable the size and divergence of the X-ray beam to be altered readily to match the different requirements of successive samples and appropriate designs are proposed. Alternative types of mirror are expected to be advantageous, especially for the smallest crystals. For crystals of sizes 300 microm or less, which need a small well collimated beam with low divergence, the output from this X-ray tube running at 24 W provides a usable flux similar to that available from rotating-anode generators. The relative performance of this tube and mirror combination becomes increasingly advantageous with the study of ever-smaller crystals.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Cristalografía por Rayos X/instrumentación , Fragmentos Fab de Inmunoglobulinas/química , Óptica y Fotónica , Programas InformáticosRESUMEN
In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.
Asunto(s)
Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Proteoma/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Electroforesis en Gel Bidimensional , Genoma Bacteriano , Mycobacterium tuberculosis/patogenicidad , Especificidad de la Especie , Virulencia/genéticaRESUMEN
A variety of different devices has been described recently for conditioning the X-ray beam incident on the sample for structural studies on proteins and other macromolecular crystals.
Asunto(s)
Cristalografía por Rayos X/instrumentación , Cristalografía por Rayos X/métodos , Proteínas/química , Diseño de Equipo , Óptica y FotónicaRESUMEN
Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.
Asunto(s)
Bases de Datos Factuales , Electroforesis en Gel Bidimensional , Internet , Mycobacterium bovis/química , Mycobacterium tuberculosis/química , Proteoma , Proteínas Bacterianas/análisis , Electroforesis en Gel Bidimensional/métodosRESUMEN
In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.
Asunto(s)
Cardiopatías/genética , Infecciones/genética , Neoplasias/genética , Proteínas/genética , Animales , Antígenos/análisis , Borrelia/inmunología , Electroforesis en Gel Bidimensional , Femenino , Humanos , Embarazo , Toxoplasma/inmunologíaRESUMEN
The Tradescantia-stamen hair (Trad-SHM) and -micronucleus (Trad-MCN) bioassays were used to determine the genotoxicity of two eluates derived from mine tailings. The goal was to test the suitability of the Tradescantia bioassays as screening tools for this kind of waste material. Leachates obtained using the current standard German leaching test methods (S4 eluate) as well as leachates obtained using a new eluation method (pHstat4) were tested and compared. Concentration of heavy metals in the pHstat4 eluate were much higher than in the S4 eluate. The chemical analysis corresponded well with the results of the bioassays. Exposure to solutions containing more than 1% pHstat4 eluate caused a significantly higher number of micronuclei. The Trad-SHM bioassay also showed an increased pink mutation rate when plants were exposed to 8 or 16% eluate solutions. In contrast, the S4 eluate only caused increased mutation rates when solutions containing more than 32% eluate were used. The low pH of the pHstat4 eluate was not responsible for the genotoxicity observed using both bioassays, as indicated by the lack of significant mutation rates in the nitric acid controls. This demonstrates that the Tradescantia bioassays can be used as tools to assess the genotoxic potential of environmental samples with a wide range of pH values, without the need for sample modification.
Asunto(s)
Residuos Industriales , Metales Pesados/toxicidad , Minería , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Plantas/efectos de los fármacos , Bioensayo , Monitoreo del Ambiente/métodos , Alemania , Metales Pesados/análisis , Pruebas de Micronúcleos , Plantas/genética , Salud UrbanaRESUMEN
Cardiac hypertrophy and heart failure are frequently accompanied by elevated plasma levels of tumor necrosis factor alpha (TNF alpha), the pathogenetic relevance of this finding being a matter of debate. In human acute septic cardiomyopathy, on the other hand, the negative inotropic impact of TNF alpha on the heart is well documented and frequently ascribed to the induction of inducible nitric oxide (NO) synthase (iNOS) and an enhanced production of NO in the heart. Yet the present study presents evidence that in cardiomyocytes TNF alpha in non-toxic concentrations specifically depresses contractile performance independent of NO. In spontaneously beating neonatal rat cardiomyocytes, TNF alpha in a low, pathophysiologically relevant concentration (10 U/ml, 1-3 days) does not alter basal pulsation amplitude, but blocks alpha- and beta-adrenoceptor-stimulated increase in contractility and beating irregularity and impairs the impact of high extracellular calcium on contractile performance. However, this low TNF alpha-concentration does not suffice to induce iNOS - documented by reverse transcriptase polymerase chain reaction - or enhance nitrite concentrations in the cell culture supernatants as a measure of cellular NO production, neither in the presence nor absence of dexamethasone (0.1 micro M). Only in high concentration - the specific proinflammatory action being documented by an enhanced release of interleukin-6 from cardiomyocytes - TNF alpha (1000 U/mol; 6, 24 h) weakly induces the mRNA for iNOS, with a consecutive moderate rise in cellular nitrite production. TNF alpha-incubation (10-1000 U/ml) does not alter the morphological appearance of the cells displayed by phase contrast microscopy or evoke gross cytotoxicity.
Asunto(s)
Contracción Miocárdica/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Depresión Química , Inducción Enzimática , Humanos , Interleucina-6/metabolismo , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacologíaRESUMEN
The master gel of the human myocardial two-dimensional electrophoresis (2-DE) gel database contains about 3300 protein spots characterized in terms of isoelectric point (pI) and molecular mass. A high-performance technique was applied, using large gels (23 x 30 cm). Isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) was combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 15% acrylamide gels in the second dimension. The range of pI extends from pH 4.5 to 9.6. Seventy proteins were identified by combinations of amino acid analysis, N-terminal and internal sequencing, immunostaining, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting, post-source decay MALDI-MS and ladder sequencing by carboxypeptidase P. The identification of additional proteins, not found in the master gel, was achieved by immunoblotting. Unequivocal identification with high sensitivity and good yield was obtained by combining internal sequencing and MALDI-MS. In-gel digestion, the concentration and purification of peptides in a peptide collecting device, and the improved FRAGMOD program for peptide mass fingerprinting have added to the security and sensitivity of identification. The high-performance human myocardial 2-DE database was built up with proteins detected by the TOPSPOT program. Spots within six sections of the whole pattern are clickable. Protein description includes detailed information about identification, characterization, and links to the related SWISS-PROT, other 2-DE databases and Medline entries. The database is constructed in accordance with four of the rules for a federated database.