RESUMEN
KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.
Asunto(s)
Channelrhodopsins , Rhinosporidium , Humanos , Channelrhodopsins/química , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Channelrhodopsins/ultraestructura , Microscopía por Crioelectrón , Canales Iónicos , Potasio/metabolismo , Rhinosporidium/químicaRESUMEN
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Asunto(s)
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Activación del Canal Iónico , Animales , Channelrhodopsins/ultraestructura , Microscopía por Crioelectrón , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , Optogenética , Filogenia , Ratas Sprague-Dawley , Bases de Schiff/química , Células Sf9 , Relación Estructura-ActividadRESUMEN
AIMS: The aim of this study was to evaluate differences in vulnerability to traumatic stress and the 1-year course of post-traumatic stress symptoms among patients with pre-existing psychiatric disorders after the Great East Japan Earthquake. METHODS: The Impact of Event Scale-Revised (IES-R) was used to assess post-traumatic stress symptoms in 612 patients with schizophrenic (ICD-10 F2; n = 163), mood (F3; n = 299), or neurotic disorders (F4; n = 150) at 1-4 months and again at 13-16 months after the disaster (retention rate: 68%). RESULTS: The mean IES-R total score for all diagnostic groups was 18.6 at index and 13.4 at follow up. The mean IES-R total score for patients with neurotic disorders (22.5) was significantly higher than that of patients with mood disorders (18.1) and schizophrenic disorders (15.9). At follow up, these scores decreased for all groups and inter-group differences were not observed. CONCLUSIONS: Vulnerability to traumatic stress after a disaster was most severe in patients with neurotic disorders, followed by mood disorders, and, lastly, schizophrenic disorders. This difference among the three diagnostic groups was not found 1 year after the disaster.
Asunto(s)
Desastres , Terremotos , Trastornos Mentales/epidemiología , Trastornos Mentales/psicología , Trastornos por Estrés Postraumático/epidemiología , Trastornos por Estrés Postraumático/psicología , Comorbilidad , Femenino , Estudios de Seguimiento , Humanos , Japón/epidemiología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Although the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders abandoned the use of the specifier 'late-onset', a considerable number of studies have reported clinical characteristics of late-onset schizophrenia. Still, only limited research has been conducted on late-onset schizophrenia, especially in Asian countries. In this epidemiological study, the clinical characteristics of late-onset schizophrenia were examined in comparison with early-onset schizophrenia. METHODS: All patients with schizophrenia admitted to the psychiatric ward of Jichi Medical University Hospital between 1 April 1993 and 31 March 2006 were divided into two groups according to age at first onset: ≥40 years (late-onset group) and <40 years (early-onset group). The sex ratio, presence or absence of depression, schizophrenia subtype, premorbid character, marital history, and employment history at first onset were compared between the two groups. RESULTS: Of the 316 patients with schizophrenia identified, 38 patients were assigned to the late-onset group and 278 patients to the early-onset group. Mean age at onset was 23.9 ± 8.2 years for all men and 28.0 ± 13.5 years for all women. The late-onset group was characterized by more women, more paranoid type, more depressive symptoms, less introverted premorbid character, better premorbid adaptation and less neuroleptics. CONCLUSION: The characteristics of late-onset schizophrenia in Japan are in line those reported previously.
Asunto(s)
Esquizofrenia/epidemiología , Psicología del Esquizofrénico , Adulto , Edad de Inicio , Antipsicóticos/uso terapéutico , Clorpromazina/uso terapéutico , Femenino , Humanos , Pacientes Internos/psicología , Pacientes Internos/estadística & datos numéricos , Entrevista Psicológica/métodos , Japón/epidemiología , Masculino , Servicio de Psiquiatría en Hospital , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Esquizofrenia/tratamiento farmacológico , Factores Sexuales , Conducta Social , Adulto JovenRESUMEN
BACKGROUND: The Great East Japan Earthquake and subsequent tsunami of March 11, 2011 severely damaged a widespread region of northeastern Japan. Consequently, the Fukushima Nuclear Power Plant experienced a level seven 3 reactors melted down, which released a large amount of radioactive materials into the air. Due to the structural damage and radiation leaks, the victims are facing prolonged psychological distress. METHODS: Eighty-two subjects with mental disorders who made their initial visit during the first 4 months after the earthquake and one hundred and ninety-four subjects with mental disorders who had been admitted during the first one year after the earthquake to the Jichi Medical University Hospital, which is located at the edge of the disaster-stricken region, were recruited for this study. Enrolled participants were assessed according to ICD-10. A questionnaire survey was employed to evaluate the severity of psychological distress and total amount of damage. RESULTS: The conditions of 22% of the outpatients had been worsened by the psychological distress related to the earthquake. Seven percent of the patients who had been hospitalized showed marked exacerbations due to the psychological distress associated with the disaster. COMMENTS: It is of note that the exacerbation of psychiatric symptoms due to the disaster was evident among patients with mental disorders who lived even at the edge of the disaster area (i. e., subject to an earthquake intensity of 5 upper and 150 km from the Fukushima Nuclear Power Plant). The results suggest that the close follow-up of disaster victims with mental disorders is of critical importance.
Asunto(s)
Desastres , Terremotos , Accidente Nuclear de Fukushima , Trastornos Mentales/epidemiología , Estrés Psicológico/terapia , Tsunamis , Adulto , Anciano , Femenino , Humanos , Japón , Masculino , Trastornos Mentales/psicología , Persona de Mediana Edad , Adulto JovenRESUMEN
Channelrhodopsins are microbial rhodopsins that work as light-gated ion channels. Their importance has become increasingly recognized due to their ability to control the membrane potential of specific cells in a light-dependent manner. This technology, termed optogenetics, has revolutionized neuroscience, and numerous channelrhodopsin variants have been isolated or engineered to expand the utility of optogenetics. Pump-like channelrhodopsins (PLCRs), one of the recently discovered channelrhodopsin subfamilies, have attracted broad attention due to their high sequence similarity to ion-pumping rhodopsins and their distinct properties, such as high light sensitivity and ion selectivity. In this review, we summarize the current understanding of the structure-function relationships of PLCRs and discuss the challenges and opportunities of channelrhodopsin research.
Asunto(s)
Canales Iónicos , Bombas Iónicas , Channelrhodopsins/genética , Optogenética , Rodopsina/metabolismo , LuzRESUMEN
We have previously demonstrated that ischemia caused by acute myocardial infarction induces an abrupt increase of serum deoxyribonuclease I (DNase I) activity. In this study, we examined whether hypoxia can affect the levels of DNase I activity and/or its transcripts in vitro. We first exposed the human pancreatic cancer cell line QGP-1, which is the first documented DNase-I-producing cell line, to hypoxia (2% O2), and found that this induced a significant increase in both the activity and transcripts of DNase I. This response was mediated by increased transcription only from exon 1a of the two alternative transcription-initiating exons utilized simultaneously in the human DNase I gene (DNASE1); exposure of QGP-1 cells to hypoxia for 24 h resulted in a 15-fold increase of DNASE1 transcripts starting from exon 1a compared with the expression level under normoxic conditions. Promoter, electrophoretic mobility shift, and chromatin immunoprecipitation assays with QGP-1 cells exposed to hypoxia or normoxia showed that the region just upstream from exon 1a was involved in this response in a hypoxia-induced factor-1-independent, but at least in a Sp1 transcription factor-dependent manner possibly through enhanced binding of Sp1 protein to the promoter. These results indicate that DNASE1 expression is upregulated by hypoxia in the cells.
Asunto(s)
Desoxirribonucleasa I/biosíntesis , Desoxirribonucleasa I/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Hipoxia/genética , Neoplasias Pancreáticas/genética , Regulación hacia Arriba/genética , Línea Celular Tumoral , Humanos , Hipoxia/enzimología , Hipoxia/metabolismo , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/fisiologíaRESUMEN
Ugl-Y (young age-related urinary glycoprotein) is an age-specific protein that we have previously identified in urine from healthy subjects under 18 years of age. Isoelectric focusing analysis of Ugl-Y gives a set of three bands, Y1, Y2 and Y3, in the pH region around 3. To determine the complete structure of Ugl-Y, purified Y1 and Y2 from pediatric urine were enzymatically cleaved, and the resulting peptides were analyzed by protein sequencing and/or MALDI-TOF MS. As a result, it was demonstrated that Y1 consists of 189 amino acid residues, and is identical to the region from A723 to R911 of fibronectin, whereas Y2 consists of 181 amino acid residues, and is identical to the region from A723 to R903. Electrophoretic analysis of the lysate prepared from COS-7 cells transfected with Y1- or Y2-expressing vectors gave specific bands corresponding to Y1 or Y2, respectively, showing the validity of the sequences determined. Partial purification of pediatric serum followed by western blotting revealed that Ugl-Y is derived from plasma. Furthermore, Ugl-Y was generated by in vitro digestion of fibronectin by acid protease in extracts of osteoclast cells. These findings suggest that Ugl-Y is probably produced by degradation of fibronectin comprising bone matrix during the process of vigorous bone resorption in children and adolescents. This is the first report on the identification and characterization of juvenile-specific fibronectin fragments excreted into urine.
Asunto(s)
Fibronectinas/clasificación , Fibronectinas/orina , Fragmentos de Péptidos/clasificación , Fragmentos de Péptidos/orina , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Extractos Celulares , Línea Celular , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Fibronectinas/química , Fibronectinas/genética , Regulación de la Expresión Génica , Humanos , Focalización Isoeléctrica , Masculino , Datos de Secuencia Molecular , Osteoclastos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Sensibilidad y Especificidad , Alineación de SecuenciaRESUMEN
Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.
Asunto(s)
Quimotripsina/química , Desoxirribonucleasa I/química , Desoxirribonucleasa I/genética , Páncreas/química , Glándula Parótida/química , Tripsina/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Desoxirribonucleasa I/biosíntesis , Caballos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Especificidad de Órganos , Páncreas/enzimología , Glándula Parótida/enzimología , Filogenia , Alineación de Secuencia , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site approximately 12 kb upstream of exon 1, which was previously believed to be the single transcription-starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription-starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.
Asunto(s)
Empalme Alternativo , Desoxirribonucleasa I/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/fisiología , Transcripción Genética , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Desoxirribonucleasa I/metabolismo , Exones , Humanos , Datos de Secuencia Molecular , Neoplasias Pancreáticas/genética , ARN Mensajero/metabolismo , Factor de Transcripción Sp1/metabolismoRESUMEN
Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.
Asunto(s)
Anticuerpos Monoclonales , Endodesoxirribonucleasas/inmunología , Hígado/química , Animales , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Endodesoxirribonucleasas/química , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , ConejosRESUMEN
BACKGROUND: The delayed release of serum cardiac markers such as creatine kinase isoenzyme MB and equivocal early electrocardiographic changes have hampered a diagnosis of acute myocardial infarction (AMI) in the early phase after its onset. Therefore, a reliable serum biochemical marker for the diagnosis of AMI in the very early phase is desirable. METHODS AND RESULTS: Serum samples were collected from the patients with AMI, unstable angina pectoris, stable angina pectoris, and other diseases. Levels of serum deoxyribonuclease I (DNase I) activity in the patients were determined. An abrupt elevation of serum DNase I activity was observed within approximately 3 hours of the onset of symptoms in patients with AMI, with significantly higher activity levels (21.7+/-5.10 U/L) in this group compared with the other groups with unstable angina pectoris (10.4+/-4.41 U/L), angina pectoris (10.8+/-3.70 U/L), and other diseases (9.22+/-4.16 U/L). Levels of the DNase I activity in serum then exhibited a marked time-dependent decline within 12 hours and had returned to basal levels within 24 hours. CONCLUSIONS: We suggest that serum DNase I activity could be used as a new diagnostic marker for the early detection of AMI.
Asunto(s)
Desoxirribonucleasa I/sangre , Infarto del Miocardio/diagnóstico , Adulto , Anciano , Biomarcadores/sangre , Enfermedad Coronaria/diagnóstico , Desoxirribonucleasa I/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Y-chromosomal polymorphic STRs are a powerful tool for forensic and evolutionary studies. Within the last decade, a series of Y-STR systems have been developed and demonstrated to be suitable for a variety of forensic applications including sexual assault cases and paternity testing. This review describes our recent studies on novel male-specific Y-STRs, involving identification, development of a multiplex-PCR system, population study and forensic application.
Asunto(s)
Cromosomas Humanos Y , Dermatoglifia del ADN , Secuencias Repetidas en Tándem , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Grupos Raciales/genéticaRESUMEN
This review primarily summarizes the clinical applications of deoxyribonuclease I (DNase I). Human DNase I exhibits polymorphism at both the protein and DNA level, and thus is potentially one of the best biochemical markers for forensic practice. Clinically, DNase I activity in serum can be used as a novel diagnostic marker for the early detection of acute myocardial infarction and transient myocardial ischemia. Furthermore, the DNase I gene is considered to be one of the susceptibility genes for gastric and colorectal carcinoma, and myocardial infarction. Over the last decade since the discovery of the utility of its genetic polymorphism for forensic purposes, research on DNase I has expanded into clinical applications.
Asunto(s)
Desoxirribonucleasa I/genética , Neoplasias Gastrointestinales/enzimología , Isquemia Miocárdica/enzimología , Biomarcadores/sangre , Desoxirribonucleasa I/sangre , Neoplasias Gastrointestinales/genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Isquemia Miocárdica/genética , Polimorfismo GenéticoRESUMEN
A multiplex PCR system for five Y-STRs (DYS441, DYS442, DYS443, DYS444 and DYS445) has been improved to increase the probability of obtaining a DNA typing result from aged samples. Newly designed PCR primers for amplification of the DYS441 and DYS442 loci and optimization of PCR conditions enabled successful typing from blood and semen stains that had been stored for more than seven years at room temperature. Analysis of 340 Japanese males revealed 7, 5, 6, 5 and 4 alleles at the DYS441, DYS442, DYS443, DYS444 and DYS445 loci, respectively, yielding 122 haplotypes with a cumulative haplotype diversity of 0.97.
Asunto(s)
Cromosomas Humanos Y , Dermatoglifia del ADN/métodos , ADN/análisis , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetidas en Tándem , Cartilla de ADN , Frecuencia de los Genes , Haplotipos , Humanos , Japón , Masculino , Semen/química , Manejo de Especímenes , Factores de TiempoRESUMEN
Deoxyribonuclease I (DNase I) was confirmed to be expressed in the human pituitary gland, particularly the anterior lobe, at levels comparable to those in the pancreas. The DNase I activity and the amount of gene transcript present in the pituitary glands of individuals aged from 1 month to 89 years was significantly age-dependent, with an abrupt elevation after the neonatal and prepubertal periods irrespective of gender, followed by a gradual age-dependent decline in males and a marked reduction in females in their postreproductive period. This DNase I age dependence in the pituitary gland was not present in the pancreas and serum. These observations suggest that tissue-specific up-regulation of DNase I gene expression in the pituitary gland occur, possibly at the onset of puberty.
Asunto(s)
Envejecimiento/metabolismo , Desoxirribonucleasa I/genética , Expresión Génica , Hipófisis/enzimología , Pubertad/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Desoxirribonucleasa I/metabolismo , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Páncreas/enzimología , ARN MensajeroRESUMEN
A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.
Asunto(s)
Desoxirribonucleasa I/química , Proteínas de Peces/química , Hígado/enzimología , Páncreas/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carpas , Desoxirribonucleasa I/biosíntesis , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/inmunología , Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Regulación Enzimológica de la Expresión Génica , Hígado/inmunología , Datos de Secuencia Molecular , Páncreas/inmunología , FilogeniaRESUMEN
The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.
Asunto(s)
Desoxirribonucleasa I , Páncreas/enzimología , Secuencia de Aminoácidos , Animales , Células COS , Clonación Molecular , ADN Complementario/genética , Desoxirribonucleasa I/química , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/inmunología , Desoxirribonucleasa I/aislamiento & purificación , Perros , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
We devised a procedure that combines a simple extraction method, isoelectric focusing and activity staining using the dried agarose film overlay method, for deoxyribonuclease I (DNase I) typing from aged urine stains. DNase I types were determined without difficulty from urine stains kept at room temperature for 3 months or more in all of the samples tested. The amounts of urine stains required for typing after 3 months of storage were estimated to be equivalent to 60-120 microl of liquid urine. Therefore, considering that useful PCR-based DNA typing has not yet been developed for urine stains, DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains.
Asunto(s)
Desoxirribonucleasa I/genética , Desoxirribonucleasa I/orina , Cromatografía en Agarosa , Dermatoglifia del ADN/métodos , Marcadores Genéticos , Humanos , Focalización Isoeléctrica , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Factores de TiempoRESUMEN
Murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion and DNA-cast polyacrylamide gel electrophoresis were very useful screening methods for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.