RESUMEN
The increased analytical sensitivity of capillary electrophoresis detects additional irregularities that are suspicious for a monoclonal component. This is most noticeable in the beta-1-, beta-2- and gamma-globulin fractions. The causes of non-monoclonal irregularities are manifold, but are rarely reported back to the ordering physician. This article reviews the basic concepts to correctly identify irregularities, monoclonal and oligoclonal peaks by capillary electrophoresis. It then focuses on detecting and reporting typical non-monoclonal irregularities according to their electrophoresis fractions as well as their possible clinical implications.
Asunto(s)
Electroforesis de las Proteínas Sanguíneas/métodos , Proteínas Sanguíneas/análisis , Electroforesis Capilar/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Límite de Detección , Persona de Mediana Edad , Adulto JovenRESUMEN
The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated rs1 complements all rad and mms mutants available. Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested. RAD24 maps on chromosome V, close to RAD3 (1.3 cM). In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed. The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group). Properties of the mutant are discussed which hint at the control of late steps in the pathways.
Asunto(s)
Reparación del ADN , Genes Fúngicos , Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Reparación del ADN/efectos de la radiación , Epistasis Genética , Mutación , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Rayos UltravioletaRESUMEN
The delay of S-phase following treatment of yeast cells with DNA-damaging agents is an actively regulated response that requires functional RAD9 and RAD24 genes. An analysis of cell cycle arrest indicates the existence of (at least) two checkpoints for damaged DNA prior to S-phase; one at START (a G1 checkpoint characterized by pheromone sensitivity of arrested cells) and one between the CDC4- and CDC7-mediated steps (termed the G1/S checkpoint). When a dna1-1 mutant (that affects early events of replicon initiation) also carries a rad9 deletion mutation, it manifests a failure to arrest in G1/S following incubation at the restrictive temperature. This failure to execute regulated G1/S arrest is correlated with enhanced thermosensitivity of colony-forming ability. In an attempt to characterize the signal for RAD9 gene-dependent G1 and G1/S cell cycle arrest, we examined the influence of the continued presence of unexcised photoproducts. In mutants defective in nucleotide excision repair, cessation of S-phase was observed at much lower doses of UV radiation compared to excision-proficient cells. However, this response was not RAD9-dependent. We suggest that an intermediate of nucleotide excision repair, such as DNA strand breaks or single-stranded DNA tracts, is required to activate RAD9-dependent G1 and G1/S checkpoint controls.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/efectos de la radiación , Daño del ADN , Proteínas de Unión al ADN , Endodesoxirribonucleasas , Péptidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efectos de la radiación , Ciclo Celular/genética , Citometría de Flujo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fase G1/genética , Fase G1/efectos de la radiación , Eliminación de Gen , Genes Fúngicos , Genotipo , Péptidos y Proteínas de Señalización Intracelular , Factor de Apareamiento , Biosíntesis de Péptidos , Feromonas/biosíntesis , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Especificidad de la Especie , Transcripción GenéticaRESUMEN
In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G1 and G2 or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.
Asunto(s)
Antígenos Nucleares , ADN Helicasas , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Recombinación Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/inmunología , Ciclo Celular/genética , Reparación del ADN , Diploidia , Eliminación de Gen , Genes Fúngicos , Haploidia , Autoantígeno Ku , Fenotipo , Tolerancia a Radiación/genética , Saccharomyces cerevisiae/efectos de la radiaciónRESUMEN
RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase delta was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.
Asunto(s)
Adenosina Trifosfatasas/genética , ADN Helicasas/genética , Reparación del ADN , ADN de Hongos , Saccharomyces cerevisiae/genética , Secuencia de Bases , Datos de Secuencia Molecular , Mutagénesis , Ácidos Nucleicos Heterodúplex , Proteínas de Saccharomyces cerevisiae , Transcripción GenéticaRESUMEN
Two forms of alkaline phosphatase, extracted from human liver and named API1 and API3, are of high molecular mass, but API3 is the larger molecule and is membrane-bound while API1 is smaller and soluble. Enzyme kinetics are identical. It is suggested that API1 is produced from API3 by an endoprotease. We demonstrated the action of an endoprotease in human liver homogenate converting API3 into API1. In the absence of this enzyme no conversion occurred. This enzyme is active at an acidic pH (less than 6.5) in the presence of Ca.. or Mg.. -ions. It is inhibited by traces of EDTA. It is insensitive to diisopropyl fluoro-phosphate, to leupeptin and to reducing or oxidizing chemicals. At alkaline pH (8.6) its activity is rapidly destroyed. The enzyme is stable in acidic buffer. We conclude that API1 is indeed formed from API3 in the living cell by enzymatic conversion.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Isoenzimas/metabolismo , Hígado/enzimología , Péptido Hidrolasas/metabolismo , Humanos , Concentración de Iones de HidrógenoRESUMEN
Serum alkaline phosphatase isoenzymes were determined quantitatively by electrophoresis on cellulose acetate in 168 patients with rheumatic diseases subgrouped for disease activity. Median values of total alkaline phosphatase and bone isoenzyme activity, as well as frequency of patients showing pathological values, increased gradually and significantly corresponding to disease activity in rheumatoid arthritis and ankylosing spondylitis, from 0% in inactive to 90% in very active forms. Bone isoenzyme was much more sensitive than total alkaline phosphatase in moderate disease activity and was also correlated to the number of involved extravertebral joints and pain in ankylosing spondylitis. No correlation was found with stage or duration of disease, age, sex, and erythrocyte sedimentation rate. Additional to bone isoenzyme, liver isoenzymes were elevated in some patients, but with only a weak correlation with disease activity. The intestinal isoenzymes were always normal. We conclude that quantitative determination of serum alkaline phosphatase bone isoenzyme activity is a major indicator for the assessment of disease activity and therapeutic monitoring in rheumatoid arthritis and ankylosing spondylitis.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Artritis Reumatoide/enzimología , Isoenzimas/metabolismo , Fosfatasa Alcalina/sangre , Huesos/enzimología , Femenino , Humanos , Isoenzimas/sangre , Hígado/enzimología , Lupus Eritematoso Sistémico/enzimología , Masculino , Osteoartritis/enzimología , Espondilitis Anquilosante/enzimologíaRESUMEN
Physicians have to confront an enormous output of laboratory data. Normally, only a few experts manually perform the interpretation of highly specialised laboratory tests. PC Windows based 'MDI-LabLink' automates interpretation and expands traditional rule-based expert systems with flexibility and graphic illustrations of complex laboratory data. The laboratory can adjust the interpretation database to its specific needs, maintaining full control over program output. The structured input that 'MDI-LabLink' requires, supports dynamic test scheduling. It can be used to train personnel in the interpretation of laboratory test results. The program provides the clinician with a report that visualises the defect of the evaluated organ system with bitmap pictures and 3-dimensional graphics. Patient follow-ups present in tabular and graphical form. Changes in the severity of the pathobiochemical defect, induced, for example, by therapy, are monitored automatically. Applications available include interpretations of isoenzyme patterns, diagnosis of urinary proteinuria and cerebrospinal fluid analysis.
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Química Clínica/métodos , Sistemas de Información en Laboratorio Clínico , Interpretación Estadística de Datos , Presentación de Datos , Estudios de Seguimiento , Humanos , Isoenzimas , L-Lactato Deshidrogenasa/análisis , Estudios Longitudinales , Valores de ReferenciaRESUMEN
Alkaline phosphatases (AP) extracted in the presence of n-butanol from human liver are separated by affinity chromatography on phenylsepharose Cl-4B into two fractions named APII and APIIII. By repeated chromatography, APII was purified to a single enzyme entity with a specific activity of 1,684 kU/g protein. APIIII was purified to a specific activity of 535 kU/g protein. It consisted of only APIIII enzyme activity, but still contained gamma-glutamyltransferase activity. These two forms of AP are different in chromatographic and electrophoretic behaviour, APIIII being a larger molecule than APII. APII and APIIII are very similar in enzyme kinetic behavior, such as substrate activity, thermolability and sensitivity to different inhibitors. It is concluded from these experiments that multiple forms of AP in liver bear identical active centres, the difference is due to a modification of protein residue. It is possible that both are modified forms of one enzyme. Both are different from the AP isoenzyme that appears in serum in cholestatic patients.
Asunto(s)
Fosfatasa Alcalina/aislamiento & purificación , Hígado/enzimología , Fosfatasa Alcalina/antagonistas & inhibidores , Colestasis/enzimología , Cromatografía de Afinidad , Cromatografía en Gel , Cisteína/farmacología , Electroforesis en Acetato de Celulosa , Homoarginina/farmacología , Calor , Humanos , Isoenzimas/aislamiento & purificación , Cinética , Neuraminidasa , Fenilalanina/farmacología , Urea/farmacologíaRESUMEN
The measurement of urinary marker proteins is not a generally accepted laboratory practice because the results are difficult to interpret. MDI-LABLINK is software for classifying patterns of specific urinary marker proteins. The interpretations are completely user definable thanks to a specific 'pattern definition database'. Our interpretation set is based on Hofmann and Guder's work in measuring and interpreting single urinary proteins. We include additional marker proteins in order to adapt Boesken's SDS-PAGE classification. During the last 3 years, 1905 patterns were fully differentiated and identically interpreted. Firstly, the samples were classified into three patterns: normal (25.8%), predominantly glomerular (27.2%, selective, unselective, mixed, and with additional tubular proteins) and predominantly tubular (36.9%, complete/incomplete form, with additional glomerular proteins); 8.9% showed postrenal proteinuria. Secondly, glomerular selectivity measured by using urinary transferrin/IgG ratio alone correlates well with the established SI index (the ratio between IgG and transferrin clearances). Thirdly, the creatinine concentration substantiates the validity of the sample. The quality of the preanalytical phase can be improved through the ongoing education of the medical staff. Finally, measurement of urinary albumin and alpha-1-microglobulin is mandatory where kidney disease is suspected, has to be ruled out, or requires close monitoring, even when the total protein concentration is normal.
Asunto(s)
Sistemas de Administración de Bases de Datos , Urinálisis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Sistemas Especialistas , Femenino , Hematuria/orina , Humanos , Masculino , Persona de Mediana Edad , Proteinuria/orinaRESUMEN
Whereas some experimental evidence suggests that mutagenesis in yeast after treatment with DNA-damaging agents involves inducible functions, a general-acting error-prone repair activity analogous to the SOS system of Escherichia coli has not yet been demonstrated. The current literature on the problem of inducibility of mutagenic repair in yeast is reviewed with emphasis on the differences in the experimental procedures applied.
Asunto(s)
Reparación del ADN , Mutación , Saccharomyces cerevisiae/genética , Relación Dosis-Respuesta en la Radiación , Inducción Enzimática , Proteínas Fúngicas/biosíntesis , Cinética , Mutación/efectos de la radiación , Recombinación Genética/efectos de la radiación , Rayos UltravioletaRESUMEN
Synchronously dividing haploid yeast cells were UV-irradiated in various stages of the cell cycle after release from alpha-factor arrest. In confirmation of earlier results (Chanet et al., 1973), in wild-type strains G1/S phase cells were found to be the most sensitive and late S/G2 cells the most resistant. Stationary-phase (G0) cells were significantly more UV resistant than G1 cells. Strains defective in nucleotide excision repair lost enhanced resistance in the G2 phase and were most UV-sensitive in the G0 state. Reduced G2 resistance was also observed in rad6 mutants but not in rad9 mutants. After UV-irradiation in G1 phase rad9 mutant cells showed a reduced G1/S phase arrest.
Asunto(s)
Reparación del ADN , Saccharomyces cerevisiae/genética , Ciclo Celular/efectos de la radiación , ADN de Hongos/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Haploidia , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/efectos de la radiación , Rayos UltravioletaRESUMEN
Alkaline phosphatase bone isoenzyme activity in serum correlates significantly to micromorphometrically assessed parameters of bone apposition as well as resorption. Though as both processes appear to be coupled in renal osteopathy, bone isoenzyme activity seems to reflect mainly hyperosteoid states. The detection of abnormal findings is impressively better by bone isoenzyme analysis as compared with total alkaline phosphatase activity; pathological values are found even at the initial phase of osteopathy in early renal failure. The determination of alkaline phosphatase bone isoenzyme activity in serum is a sensitive and reliable diagnostic tool in assessing the beginning and degree of metabolic bone disease.
Asunto(s)
Fosfatasa Alcalina/sangre , Huesos/enzimología , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/enzimología , Isoenzimas/sangre , Adulto , Huesos/patología , Calcio/sangre , Femenino , Tasa de Filtración Glomerular , Humanos , Enfermedades Renales/complicaciones , Masculino , Persona de Mediana Edad , Hormona Paratiroidea/sangreAsunto(s)
Ciclo Celular , Daño del ADN , Saccharomyces cerevisiae/citología , Schizosaccharomyces/citología , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Ribonucleótido Reductasas/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Factores de Transcripción/metabolismoAsunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Animales , Transformación Celular Neoplásica , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Humanos , Leucemia Mieloide Aguda/genética , Mutagénesis , Recombinación Genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Xerodermia Pigmentosa/genéticaRESUMEN
Among the DNA repair mutants of the yeast Saccharomyces cerevisiae, the rad6 mutants are characterized by a highly pleiotropic phenotype. Most remarkably, these mutants are sensitive towards a variety of DNA-damaging agents and deficient in mutation induction. The RAD6 gene has been cloned and most recently, ubiquitin-conjugating activity of the Rad6 protein has been demonstrated. In this brief review, the properties of rad6 mutants are discussed in the light of these new findings. The available data hint at a connection between DNA repair, mutagenesis, chromosome structure and protein degradation.