RESUMO
The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates. In gad mice, accumulation of amyloid beta-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.
Assuntos
Deleção de Genes , Tioléster Hidrolases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/enzimologia , Modelos Animais de Doenças , Feminino , Biblioteca Gênica , Genes Recessivos , Ligação Genética , Isoenzimas , Masculino , Bulbo/enzimologia , Bulbo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Doenças Neurodegenerativas/genética , Homologia de Sequência de Aminoácidos , Testículo/enzimologia , Ubiquitina TiolesteraseRESUMO
Extracellular levels of the excitatory neurotransmitter glutamate in the nervous system are maintained by transporters that actively remove glutamate from the extracellular space. Homozygous mice deficient in GLT-1, a widely distributed astrocytic glutamate transporter, show lethal spontaneous seizures and increased susceptibility to acute cortical injury. These effects can be attributed to elevated levels of residual glutamate in the brains of these mice.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Epilepsia/metabolismo , Ácido Glutâmico/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Sistema X-AG de Transporte de Aminoácidos , Animais , Transporte Biológico , Encéfalo/patologia , Lesões Encefálicas/patologia , Eletroencefalografia , Epilepsia/patologia , Marcação de Genes , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural , Células Piramidais/patologia , Células Piramidais/fisiologia , Sinapses/metabolismo , Transmissão SinápticaRESUMO
Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase (GAA), a glycogen degrading lysosomal enzyme. GAA-deficient (AMD) Japanese quails exhibit progressive myopathy and cannot lift their wings, fly, or right themselves from the supine position (flip test). Six 4-wk-old acid maltase-deficient quails, with the clinical symptoms listed, were intravenously injected with 14 or 4.2 mg/kg of precursor form of recombinant human GAA or buffer alone every 2-3 d for 18 d (seven injections). On day 18, both high dose-treated birds (14 mg/kg) scored positive flip tests and flapped their wings, and one bird flew up more than 100 cm. GAA activity increased in most of the tissues examined. In heart and liver, glycogen levels dropped to normal and histopathology was normal. In pectoralis muscle, morphology was essentially normal, except for increased glycogen granules. In sharp contrast, sham-treated quail muscle had markedly increased glycogen granules, multi-vesicular autophagosomes, and inter- and intrafascicular fatty infiltrations. Low dose-treated birds (4.2 mg/kg) improved less biochemically and histopathologically than high dose birds, indicating a dose-dependent response. Additional experiment with intermediate doses and extended treatment (four birds, 5.7-9 mg/kg for 45 d) halted the progression of the disease. Our data is the first to show that an exogenous protein can target to muscle and produce muscle improvement. These data also suggest enzyme replacement with recombinant human GAA is a promising therapy for human Pompe disease.
Assuntos
Doenças das Aves/tratamento farmacológico , Coturnix , Glucana 1,4-alfa-Glucosidase/deficiência , Doença de Depósito de Glicogênio Tipo II/veterinária , alfa-Glucosidases/farmacologia , Animais , Doenças das Aves/metabolismo , Doenças das Aves/patologia , Doenças das Aves/fisiopatologia , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/patologia , Doença de Depósito de Glicogênio Tipo II/fisiopatologia , Humanos , Masculino , Músculos/efeitos dos fármacos , Músculos/fisiopatologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Distribuição Tecidual , alfa-Glucosidases/metabolismoRESUMO
Concentrations of bovine carbonic anhydrase isozyme VI (CA-IV) in bovine serum, saliva, normal milk, colostrum, submandibular gland, liver, and mammary gland were determined. CA-VI was purified from bovine saliva and an antibody to CA-VI was generated. The concentrations of CA-VI in the saliva (7.8 +/- 7.9 microg/ml), serum (2.1+/- 5.7 ng/ml), milk (7.9 +/- 12.1 ng/ml), submandibular gland (284.7 microg/g protein), liver (921.0 +/- 180.7 ng/g protein) and mammary gland (399.6 +/- 191.2 ng/g protein) were determined by ELISA. No seasonal change in CA-VI levels was observed in normal milk. The concentration of CA-VI in colostrum (day 1 post partum) was 119 ng/ml and decreased rapidly by 1 month following birth. Mammary gland contained much smaller amounts than the submandibular gland. CA-VI mRNA was detected in the liver and mammary gland of cow by RT-PCR. The ELISA used in this study proved to be a precise and sensitive method for determining CA-VI concentrations in saliva, serum, milk and tissue specimens from cows. The ELISA may enable the study of changes in CA-VI associated with hereditary or metabolic disorders of the salivary gland, mammary gland and liver using small samples of saliva, serum or milk.
Assuntos
Anidrases Carbônicas/análise , Bovinos/metabolismo , Leite/enzimologia , Saliva/enzimologia , Animais , Anidrases Carbônicas/sangue , Anidrases Carbônicas/genética , Colostro/enzimologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Fígado/enzimologia , Glândulas Mamárias Animais/enzimologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Glândula Submandibular/enzimologiaRESUMO
In mammals, a pair of ejaculatory ducts exists in the urethra at the seminal colliculus. The detailed anatomical structures of the distal end of the ejaculatory ducts of Sprague-Dawley rats were investigated by the computer-assisted three-dimensional reconstruction analysis using light-microscopic serial sections. A three-dimensional reconstruction revealed that in adult rats, the ejaculatory sinus pair consists of two parts: the cranial section - a compartment region composed of a fusion of the ampullary gland duct and the seminal vesicle duct, and the caudal section - a grooved region composed of a long slitlike ejaculatory ostium that extends into the urethra on both sides of the seminal colliculus. But the sphincter structure was not observed. The long axis of the compartment region was approximately 58 µm in length, and that of the groove region was approximately 495 µm. Although many epithelial glands ducts were distributed throughout the ejaculatory sinuses, the prostate and coagulation gland ducts did not open in these sinuses. The urethra was composed of transitional epithelium, while the ejaculatory sinuses were composed of single to stratified cuboidal epithelium. The ejaculatory ducts continued to the ejaculatory ostium in male adult Sprague-Dawley rat were composed of the seminal vesicle ducts received the ampullary gland ducts.
Assuntos
Ductos Ejaculatórios/anatomia & histologia , Imageamento Tridimensional/métodos , Glândulas Seminais/anatomia & histologia , Uretra/anatomia & histologia , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Acid maltase deficiency (AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human acid maltase (AM) cDNA downstream of the CAG promoter, composed of modified chicken beta-actin promoter and CMV IE enhancer (AxCANAM). Japanese quail with AMD was used for this study as an animal model for human AMD. When cultured fibroblasts from AMD quail were infected with AxCANAM, AM activity in the cells increased in proportion to the multiplicity of infection (MOI). When AxCANAM (4.5 x 10(8) PFU) was injected into unilateral superficial pectoral muscle of AMD quail, PAS staining showed that glycogenosomes disappeared and stainability of acid phosphatase was reduced in the injected area as compared with the contralateral muscle of the same birds. Biochemically, AM activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human AM protein processed to active forms. These results suggest that the human AM cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quail.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Glucana 1,4-alfa-Glucosidase/deficiência , Glucana 1,4-alfa-Glucosidase/genética , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Animais , Western Blotting , Células Cultivadas , Coturnix , Modelos Animais de Doenças , Fibroblastos , Terapia Genética , Vetores Genéticos , Glucana 1,4-alfa-Glucosidase/metabolismo , Humanos , Músculo Esquelético/patologia , Regiões Promotoras GenéticasRESUMO
Cloning and sequencing of the peripheral myelin protein-22 cDNA and genomic DNA from newly found Trembler mice revealed an in-frame deletion including exon IV which codes for the second (TM2) and a part of third (TM3) transmembrane domain of peripheral myelin protein-22. This mutation was distinct from those in both other allelic Trembler and Trembler-J mice, which carry point mutations within the putative transmembrane spanning regions of peripheral myelin protein-22. Inheritance was autosomal dominant. The affected mice revealed an abnormal gait, which appeared at 15-20 days of age, followed by motor and sensory ataxia, which remained throughout life. Most of the affected mice could survive more than one year. One of the most notable pathological phenotypes was a giant vacuolar formation in the sciatic nerve of homozygotes. They vary in size within the cytoplasm of Schwann cells, which failed to assemble myelin at any ages studied. Heterozygotes showed normal myelination during the early postnatal stages, followed by a segmental demyelination at an advanced stage. Vacuolar formation was not so frequent as in the homozygotes. These results suggest that the missing of transmembrane spanning region (TM2 and TM3) of peripheral myelin protein-22 may disturb a dual biological function of peripheral myelin protein-22, leading to a dysmyelination of axons and to a vacuolar formation within the cytoplasm of the Schwann cells. The latter phenotype is discussed in conjunction with the disruption of an intracellular transport system and subsequent cell death.
Assuntos
Morte Celular/fisiologia , Deleção de Genes , Proteínas da Mielina/genética , Células de Schwann/metabolismo , Nervo Isquiático/ultraestrutura , Animais , Sequência de Bases , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Dados de Sequência Molecular , Nervo Isquiático/metabolismoRESUMO
We separated two forms of arylalkylamine N-acetyltransferase (AANAT) from various organs of the American cockroach, Periplaneta americana. Both forms of the enzyme had an equivalent molecular mass of 28 kDa. One form isolated from the testicular accessory glands had high enzyme activity at acidic pHs. The isoelectric point was 5-6 and the substrate specificity was wider than the other type. The other isolated form from female midguts had a higher level of enzyme activity at basic pHs. These findings suggested that P. americana contains polymorphic AANAT, as is the case in Drosophila melanogaster. These forms differed not only in pH specificity, and substrate specificity but in chromatographic behavior and kinetic properties. Most of the organs we examined contained a mixture of the two forms since two types of AANAT activity were separated in different chromatographic fractions when two pH conditions were used for activity measurement.
Assuntos
Arilamina N-Acetiltransferase/metabolismo , Periplaneta/enzimologia , Polimorfismo Genético , Animais , Arilamina N-Acetiltransferase/isolamento & purificação , Cromatografia Líquida/métodos , Sistema Digestório/enzimologia , Feminino , Cinética , Masculino , Periplaneta/genética , Especificidade por SubstratoRESUMO
Arylalkylamine N-acetyltransferase (NAT; EC2.3.1.87) catalyzes N-acetylation of various arylalkylamines using acetyl-CoA as a donor substrate. A type of NAT was purified 2700-fold from 451 pairs of cockroach testicular organs consisting of testis and its accessory gland. The NAT activity was recovered as a single peak on any column chromatography examined, suggesting that the testicular organ contained only one form of NAT. Five steps of successive column chromatographies gave a single protein band on SDS-polyacrylamide gel electrophoresis with estimated molecular mass of 28 kDa. The molecular mass of the native enzyme was also determined to be approximately 30 kDa by molecular sieve chromatography, indicating that the enzyme is a monomer protein. The enzyme acted on various arylalkylamines such as tryptamine, serotonin, dopamine, octopamine, norepinephrine, tyramine and methoxytryptamine, with K(m) values ranging from 20 to 50 microM. The optimum pH for these substrates was around 6.0. Internal amino acid sequences derived from two proteolytic fragments of the enzyme were determined as Leu-Leu-Gly-Glu-Asn-Gly-Asp-Glu and Phe-Phe-Phe-Leu-Glu-Glu-Pro-Leu-Asn-Ile-Ser-Leu-Gln, both of which exhibited significant homology to the C-terminal sequence of known vertebrate NATs; however, homology was less than 45%. These results suggest that a unique NAT is present in the cockroach testicular organ at high levels, and likely plays a role in the regulation of testicular function.
Assuntos
Arilamina N-Acetiltransferase/química , Periplaneta/enzimologia , Sequência de Aminoácidos , Animais , Arilamina N-Acetiltransferase/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Masculino , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Testículo/enzimologiaRESUMO
The significance of the route for administration of murine recombinant interferon-beta (IFN-beta) for inducing its therapeutic effects has been studied. BALB/c mice were daily injected intravenously, intramuscularly or subcutaneously with 1.5x10(3), 1. 5x10(4), or 1.5x10(5) IU of IFN-beta, from one day before to 8th day after mouse hepatitis virus (MHV-2) challenge. All mice received IFN-beta survived significantly longer than those without IFN. In the liver of those IFN-treated mice, viral growth and the histopathological damages were extremely alleviated. These results suggest that, irrespective of the differences in the route of administration, IFN-beta markedly suppressed viral activity when its administration was started prior to viral infection. For clinical use, however, further studies are needed on the optimal route for administration if IFN-beta is given after viral infection.
Assuntos
Infecções por Coronaviridae/tratamento farmacológico , Hepatite Viral Animal/tratamento farmacológico , Injeções Intramusculares , Injeções Intravenosas , Injeções Subcutâneas , Interferon Tipo I/uso terapêutico , Animais , Infecções por Coronaviridae/patologia , Infecções por Coronaviridae/virologia , Hepatite Viral Animal/patologia , Hepatite Viral Animal/virologia , Interferon Tipo I/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas RecombinantesRESUMO
The occurrence and formation of UDP-N-acetyl-D-glucosaminuronic acid (UDP-GlcNAcA) and UDP-N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA) were studied in Micrococcus luteus ATCC 4698. UDP-N-acetylhexosaminuronic acid separated from D-cycloserine-inhibited cells was shown to be a mixture of UDP-GlcNAcA and UDP-ManNAcA in the ratio of 87:13, whereas that obtained from untreated cells was a 96:4 mixture of these two nucleotides. Crude enzyme preparations obtained from the supernatant fraction of cells catalyzed the NAD+-dependent conversion of UDP-GlcNAc into UDP-GlcNAcA and UDP-ManNAcA. Studies on the partial separation and properties of enzymes revealed that UDP-GlcNAcA is synthesized directly from UDP-GlcNAc by the action of UDP-GlcNAc dehydrogenase and that UDP-ManNAcA is synthesized from UDP-GlcNAc through the successive actions of UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase. However, enzymatic conversion of UDP-GlcNAcA to UDP-ManNAcA was not detected. Ammonium sulfate protects both dehydrogenases from inactivation during storage and incubation. Partially purified UDP-GlcNAc dehydrogenase required dithiothreitol and the particulate fraction for its full activity. The apparent Km values of UDP-GlcNAc dehydrogenase for UDP-GlcNAc and NAD+ were 0.28 and 1.43 mM, respectively. The optimum pH of this enzyme was higher than 9 in Tris-HCl buffer. p-Chloromercuribenzoate at 27 microM as well as 10 mM ethanol almost completely inhibited the UDP-GlcNAc dehydrogenase reaction.
Assuntos
Micrococcus/metabolismo , Açúcares de Uridina Difosfato/biossíntese , Desidrogenases de Carboidrato/metabolismo , Isomerismo , Cinética , Micrococcus/enzimologia , NAD/metabolismoRESUMO
The GAD mouse is a spontaneous neurological mutant with axonal dystrophy in the gracile tract of the medulla oblongata and spinal cord. The immunoreactivity of amyloid precursor protein (APP-IR) and amyloid beta-protein (A beta P-IR) was examined in the gracile tract and the dorsal root ganglia of normal and GAD mice. The mice were studied at 4, 9, 18, and 32 weeks of age. These periods correspond clinically to the initial, progressive, critical, and terminal stages of the disease, respectively. The APP-IR in both axons and glial cells was already accentuated to a higher level as early as 4 weeks of age in the gracile nucleus of GAD mouse. Similarly there was increase in APP-IR of GAD mouse in the dorsal root ganglia. Almost all of the primary neurons in the dorsal root ganglia at the lumbar cord level of GAD mouse revealed stronger APP-IR than those of normal mouse throughout all stages. The cells showing immunoreactivity for amyloid beta-protein became positive in axons and glial cells in the gracile nucleus by approximately the 9th week, and followed by an increase of A beta P-IR in order of the cervical, thoracic and lumbar spinal cords. These results suggest that the initial feature in GAD mouse is an accumulation of amyloid precursor protein induced by axonal dystrophy which then leads to a deposition of amyloid beta-protein within the cytoplasm of both axons and glial cells in the gracile tract.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Axônios/fisiologia , Bulbo/metabolismo , Degeneração Neural/fisiologia , Doenças Neuromusculares/metabolismo , Medula Espinal/metabolismo , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Imuno-Histoquímica , Bulbo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Vias Neurais/metabolismo , Vias Neurais/patologia , Doenças Neuromusculares/genética , Medula Espinal/patologiaRESUMO
To evaluate the excitabilities of the transcortical loop in patients with dentatorubral-pallidoluysian atrophy (DRPLA), we studied somatosensory evoked potentials (SEPs) and evoked EMG responses (V1 and V2) in 10 patients and age-matched controls. In addition, the facilitatory effects of somatosensory inputs on motor evoked potentials (MEPs) were studied in four patients and controls. We observed attenuated or prolonged cervical and subcortical potentials and prolonged middle latency components of SEPs. The amplitudes of V2 in patients were significantly lowered compared to those in the controls, while the amplitudes and latencies of V1 were similar between the two groups. Since V2 was considered as a transcortical reflex, our results suggest reduced excitabilities of the afferent pathway of the transcortical loop in patients with DRPLA. Median nerve stimulation (MNS) 25 to 30 ms preceding transcranial magnetic stimulation (TMS) facilitated MEPs in the thenar muscle in two of the four patients and in the controls. The facilitation of MEPs by MNS tended to be independent of the reduction in V2. Such a result suggests that different neural mechanisms elicit V2 and facilitate MEPs following peripheral nerve stimulation, although further studies are needed. The combination of SEPs, evoked EMG responses and MEPs may be a useful technique to detect abnormalities of input and output coordinations of the transcortical loop.
Assuntos
Potencial Evocado Motor/fisiologia , Potenciais Somatossensoriais Evocados/fisiologia , Epilepsias Mioclônicas Progressivas/fisiopatologia , Adolescente , Adulto , Estimulação Elétrica , Eletromiografia , Humanos , Magnetismo , Análise por Pareamento , Nervo Mediano/fisiologia , Pessoa de Meia-Idade , Epilepsias Mioclônicas Progressivas/genética , Reflexo Anormal/fisiologiaRESUMO
AIM: To determine if the diuretic spironolactone cross reacts with 17alpha-hydroxyprogesterone (17OHP) in an enzyme linked immunosorbent assay (ELISA) kit used for the mass screening of congenital adrenal hyperplasia. METHODS: Concentrations of 17OHP on a blood filter paper disc were measured using an ELISA kit (kit C-7: ENZAPLATE N-17alpha -OHP-7; Chiron, Tokyo, Japan). The cross reactivity of spironolactone and its metabolites with 17OHP was determined. The concentrations of spironolactone and its metabolites in blood were measured using HPLC (high performance liquid chromatography). RESULTS: Spironolactone cross reacted with 17OHP using kit C-7 (0.01%), by increasing 17OHP concentration in a dose dependent manner. The blood concentration of spironolactone and its metabolites was nearly 900 ng/ml, high enough to show an additive effect on the 17OHP concentration. About 12% of the false positive cases screened using the kit were due to the administration of spironolactone. CONCLUSIONS: Spironolactone interferes with 17OHP concentrations, leading to false positive test results for CAH.
Assuntos
17-alfa-Hidroxiprogesterona/metabolismo , Hiperplasia Suprarrenal Congênita/diagnóstico , Diuréticos/metabolismo , Ensaio de Imunoadsorção Enzimática , Espironolactona/metabolismo , Hiperplasia Suprarrenal Congênita/sangue , Reações Falso-Positivas , Humanos , Recém-Nascido , Masculino , Triagem Neonatal/métodosRESUMO
Localizations of carbonic anhydrase isoenzymes (CA I, CA II and CA III) were investigated immunohistochemically in the salivary glands and intestine of mature and suckling pigs. Carbonic anhydrase isoenzymes were not detected in the salivary glands of sucklings, but were present in the adult. Bicarbonate ion in saliva might be important for the digestion of solid foods in mature pigs, but unnecessary for the digestion of milk in sucklings. Expressions of CA I and CA II were detected strongly in the large intestine of the adult and sucklings, and faintly only at duodenum in the small intestine. CA I and CA II isoenzymes in the large intestine may be involved, at least in part, in ion absorption and water metabolism during digestion and absorption of milk in suckling pigs. In addition, CA I and CA II expression in the duodenal villus enterocyte may support the process of bicarbonate absorption in the duodenum.
Assuntos
Anidrases Carbônicas/metabolismo , Intestino Grosso/enzimologia , Intestino Delgado/enzimologia , Glândulas Salivares/enzimologia , Suínos/anatomia & histologia , Fatores Etários , Animais , Animais Lactentes , Western Blotting/veterinária , Anidrases Carbônicas/biossíntese , Feminino , Imuno-Histoquímica/veterinária , Isoenzimas/biossíntese , Isoenzimas/metabolismo , MasculinoRESUMO
OBJECTIVE: To elucidate locations of cytosolic carbonic anhydrase isoenzyme (CA-I, CA-II, and CA-III)-positive epithelial cells in equine male reproductive organs. DESIGN: Descriptive and immunohistochemical study. ANIMALS: 4 clinically normal male horses. PROCEDURE: The testis (seminiferous tubules, rete tubules), epididymis (initial, middle, and terminal segments), proximal and distal portions of the ductus deferens, ampulla ductus deferentis, seminal vesicle, prostate, and bulbourethral gland were excised from euthanatized horses after administration of an overdose of pentobarbital. The tissue specimens were quickly placed in fixative solution, dehydrated in ethanol, and embedded; then thin sections were cut. For immunohistochemical staining, antibodies against purified equine CA-I, CA-II, and CA-III were raised in rabbits. After examination of the specificity of each antiserum, the monospecific antisera against carbonic anhydrase isoenzymes were used to localize the isoenzymes. RESULTS: Specific staining for CA-III was found in the Sertoli and basal cells of the ductus deferens. Most of the testicular and epididymal tissue, as well as ductus deferens, were virtually negative for the enzymes when stained with the antibody to CA-I and CA-II. In the initial segment of the epididymis, a few principal cells had intense cytoplasmic staining with anti-CA-II. In the male accessory glands, CA-I, CA-II, and CA-III were detected in the epithelial cells of the seminal vesicle, prostate, and bulbourethral gland. CONCLUSIONS: In the equine male reproductive tract, the bicarbonate in semen originates mainly from accessory reproductive glands. All 3 isoenzymes may have central roles in the regulation of bicarbonate concentration in seminal plasm and, accordingly, regulate seminal plasma pH. Distribution of CA-III in Sertoli and basal cells of the ductus deferens suggests other specialized physiologic roles.
Assuntos
Anidrases Carbônicas/análise , Genitália Masculina/enzimologia , Isoenzimas/análise , Animais , Anticorpos , Glândulas Bulbouretrais/citologia , Glândulas Bulbouretrais/enzimologia , Epididimo/citologia , Epididimo/enzimologia , Genitália Masculina/anatomia & histologia , Genitália Masculina/citologia , Cavalos , Imuno-Histoquímica/métodos , Masculino , Próstata/citologia , Próstata/enzimologia , Coelhos , Glândulas Seminais/citologia , Glândulas Seminais/enzimologia , Testículo/citologia , Testículo/enzimologia , Ducto Deferente/citologia , Ducto Deferente/enzimologiaRESUMO
Japan was the first country to establish a nationwide quality control system. When the Japanese Federal Government initiated Nationwide Neonatal Screening in 1977, the system officially included a Quality Control (QC) System that should cover all screening laboratories in Japan. This QC system is quite different from that for usual clinical chemistry. The aim of the National QC System for Neonatal Screening is evaluation of the accuracy of the tests and evaluation of the ability to detect suspicious samples with very mild abnormalities. For accomplishing the aim, the QC center established an inter-laboratory QC survey Screening laboratories having weak points can be identified through the inter-laboratory QC survey, and the Center must find a way to improve the ability of these screening laboratories. This requires a nationwide consensus regarding the cut-off levels of tested materials. Based on the cooperation of the Societies For Mass-screening, of Inborn Errors of Metabolism and of Pediatric Endocrinology, we set low cutoff levels for each compound to minimize the number of false negative cases. The system also included the evaluation of the quality of essential screening reagents and the special filter paper for blood collection (in partnership with the production companies). For this purpose, we developed some new methods for evaluating the standard-compounds for the various screening tests exactly, except in the case of TSH screening.
Assuntos
Laboratórios/normas , Erros Inatos do Metabolismo/diagnóstico , Programas Nacionais de Saúde/normas , Triagem Neonatal/normas , Controle de Qualidade , Gestão da Qualidade Total , Humanos , Recém-Nascido , JapãoRESUMO
We assessed the oropharyngeal swallowing ability in 8 patients with Parkinson's disease (PD), 8 patients with progressive supranuclear palsy (PSP), and 10 age-matched healthy controls (CTL) using videofluorography (VF). In VF studies, PD and PSP patients demonstrated food pooling on the tongue, difficulty in bolus formation, and bolus falling into pharynx before swallow. PSP patients had a significantly longer delay in the pharyngeal phase and showed food falling into larynx more often than PD patients (p < 0.05). On measurement of swallowing time periods as proposed by Robbins et al., both patient groups showed significantly longer periods during many swallowing phases (P < 0.05) compared to those in the control group, but there were no significant differences between the PD and PSP groups. However, in PSP patients, the time for "transferring the food bolus from the oral cavity to pharynx" which we defined as a distinct stage was significantly longer (p < 0.05) than that in the PD group. We think that the difference in dysphagia characteristics between the two diseases arises from the variations in pathological changes in PSP, including those in the cerebral cortex, cerebellum, pons and medulla tegmentum in addition to the basal ganglia. Dystonia in the neck muscle also plays a role in dysphagia in PSP patients. Levodopa medication, changing the form of foods and training in rehabilitation techniques such as the chin down posture, supraglottic swallowing and ice-massage of the oral region are probably effective for dysphagia in PD patients. In patients with PSP, there are few research reports about the treatment of dysphagia. However, several dysphagia treatments seem to be useful depending on the abnormal patterns in the VF. Further studies are necessary to establish more effective treatments for dysphagia in PD and PSP.
Assuntos
Transtornos de Deglutição/diagnóstico , Doença de Parkinson/complicações , Paralisia Supranuclear Progressiva/complicações , Idoso , Idoso de 80 Anos ou mais , Feminino , Fluoroscopia , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/fisiopatologia , Paralisia Supranuclear Progressiva/fisiopatologia , Gravação em VídeoRESUMO
A 71-year-old man developed dysarthria and difficulty of swallowing in December 1997. He was diagnosed as having the bulbar type of amyotrophic lateral sclerosis (ALS). In November 1998, he was admitted to our hospital to undergo treatment for bulbar palsy and respiratory discomfort. In January 1999, ventilatory support (synchronous intermittent mandatory ventilation) during sleep at night was initiated. Severe progressive hypotension and loss of consciousness were observed soon after the start of artificial respiration, and both symptoms disappeared after artificial respiration was discontinued. This phenomenon was observed consistently during ventilatory support, while unpleasant stimuli such as bronchoscopy and replacement of the cannula tube induced severe hypertension. To clarify the mechanism of underlying these abnormal changes in blood pressure, autonomic function tests were performed while awake during the daytime. Ventilatory support induced a drop in blood pressure accompanied by a decrease in influx speed to the right ventriculum, the latter of which suggested a reduction in venous return. These values returned to the baseline following detachment of the ventilator. A 60 degrees head-up tilt (HUT) angle and standing from a supine position produced orthostatic hypotension, the latter of which was accompanied by a compensatory increase in pulse rate. The basal supine plasma noradrenaline (NA) level was high and the HUT showed a slight elevation of NA. The basal supine plasma arginine vasopressin (AVP) level was within the normal range, whereas the AVP level did not increase during HUT. Urinary secretion rates of NA and 3-methoxy-4-hydroxy-phenylglycol were elevated. A cold pressor test demonstrated reflex hypertension. The oculovagal reflex, coefficient of variation of R-R intervals. (CVR-R) and increase in pulse rate in response to atropine administration were within the normal range. The combination of midodrine, L-dihydroxyphenylserine (DOPS) and increasing intravascular volume via continuous intravenous drip infusion relieved the circulatory collapse during artificial respiration. In conclusion, the present case of ALS had sympathetic hyperactivity, somatosympathetic reflex and dysregulation of the baroreflex arc. Degeneration of central autonomic network, including the hypothalamus and the central nucleus of the amygdala, which has been shown in some ALS patients, might underlie the autonomic abnormalities in this patient.
Assuntos
Esclerose Lateral Amiotrófica/complicações , Respiração Artificial , Choque/etiologia , Idoso , Esclerose Lateral Amiotrófica/fisiopatologia , Sistema Nervoso Autônomo/fisiopatologia , Humanos , Hipotensão Ortostática/etiologia , MasculinoRESUMO
Immunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in bovine male reproductive tracts were studied. In bulls, no immunoreaction was seen after treatment with antibodies to CA-I, -II and CA-III in the testis. Specific staining for CA-III, however, was evident in peritubular cells in interstitial tissue of the testis, epididymis. CA-II activity could be detected in epithelium of the epididymis, ductus deferentis and ampulla ductus deferentis. Especially, a strong reaction for CA-II was seen in apical in epithelium of the epididymis in the initial and middle segment. CA-I activity was only founded in ductus deferentis and ampulla ductus deferentis. No or a weak reaction for CA-I, CA-II and CA-III were seen in the three accessory reproductive glands. Specific immunostaining for CA-II and CA-I could be observed in the organ, suggesting the bicarbonate in bovine semen to derive primarily from the genital tract and not accessory reproductive organs. CA-III-positive peritubular cells in interstitial tissue were also stained for alpha smooth muscle actin, and were very similar to contractile myofibroblast cells (Wrobel et al., 1979).