RESUMO
Perihepatic lymph node enlargement (PLNE) which has been shown to be negatively associated with hepatocellular carcinoma (HCC) occurrence is frequently observed in chronic liver disease; however, changes in the state of perihepatic lymph nodes after eradication of hepatitis C virus (HCV) have not been investigated yet. We aimed to evaluate this issue. We enrolled 472 patients with chronic HCV infection who achieved viral eradication with direct-acting antivirals (DAA). We investigated whether the status of perihepatic lymph nodes changed before and after HCV eradication (primary endpoint). We also evaluated the association between PLNE and clinical findings such as liver fibrosis or hepatocellular injury before HCV eradication (secondary endpoint). Perihepatic lymph node enlargement was detected in 164 of 472 (34.7%) patients before DAA treatment. Surprisingly, disappearance of PLNE was observed in 23.8% (39 patients) of all PLNE-positive patients after eradication of HCV. Disappearance of PLNE was not associated with baseline clinical parameters or changing rates of clinical findings before and after DAA treatment. At baseline, presence of PLNE was significantly associated with a lower serum HCV-RNA level (P = .03), a higher serum AST level (P = .004) and a higher ALT level (P < .001) after adjustment for sex and age. In conclusion, PLNEs became undetectable after DAA treatment in 23.8% of PLNE-positive patients. Further study with a longer follow-up period is needed to clarify the clinical importance of this phenomenon especially in relationship with the risk of HCC development.
Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/patologia , Linfonodos/patologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Termite castes are a key example of polyphenism, in which reproductive division of labour is clearly seen in colonies. The reproductive castes in termites include primary and neotenic reproductives; primary reproductives found a new colony whereas neotenics succeed them in the reproductive role when the primary reproductives die or become senescent. Neotenics usually differentiate from nymphs or workers by developing functional gonads while retaining juvenile characteristics; however, the developmental mechanism during neotenic differentiation remains poorly understood. Juvenile hormone (JH) mediates a number of aspects of developmental regulation in caste differentiation in termites. In the present study we quantified JH titres in neotenic reproductives of Reticulitermes speratus, and compared these with other developmental stages. In addition, expression changes in JH signalling gene homologues (Methoprene-tolerant [Met], Krüppel-homolog1, Broad-Complex) in the head, thorax and abdomen were investigated during neotenic differentiation. Finally, we examined the function of Met in reproduction of neotenics by RNA interference (RNAi). Our results showed that the JH titres of neotenics were significantly higher than those of nymphs and workers. JH signalling genes were highly expressed in neotenic abdomens, compared with those in workers and nymphs. Met RNAi resulted in the inhibition of vitellogenin gene expression in newly moulted neotenics. These results suggest that the fertility of neotenics might be controlled by a large increase of JH titres and body-part-specific activation of JH signalling pathways.
Assuntos
Isópteros/fisiologia , Hormônios Juvenis/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Isópteros/crescimento & desenvolvimento , Muda , Ninfa/fisiologia , Interferência de RNA , Reprodução , Transdução de Sinais , Vitelogeninas/biossínteseRESUMO
Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.
Assuntos
Afídeos/metabolismo , Sesquiterpenos/metabolismo , Animais , Afídeos/genética , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Feminino , Masculino , Partenogênese , FotoperíodoRESUMO
Magneto-optic Kerr microscopy was employed to investigate the spin-orbit interactions of electrons traveling in semiconductor quantum wells using surface acoustic waves (SAWs). Two-dimensional images of the spin flow induced by SAWs exhibit anisotropic spin precession behaviors caused by the coexistence of different types of spin-orbit interactions. The dependence of spin-orbit effective magnetic fields on SAW intensity indicates the existence of acoustically controllable spin-orbit interactions resulting from the strain and Rashba contributions induced by the SAWs.
RESUMO
Bacterial biofilm formation can be induced by antimicrobial and DNA damage agents. These agents trigger the SOS response, in which SOS sensor RecA stimulates auto-cleavage of repressor LexA. These observations lead to a hypothesis of a connection between stress-inducible biofilm formation and the RecA-LexA interplay. To test this hypothesis, three biofilm assays were conducted, viz. the standard 96-well assay, confocal laser scanning microscopy, and the newly developed biofilm-on-paper assay. It was found that biofilm stimulation by the DNA replication inhibitor hydroxyurea was dependent on RecA and appeared repressed by the non-cleavable LexA of Pseudomonas aeruginosa. Surprisingly, deletion of lexA led to reduction of both normal and stress-inducible biofilm formation, suggesting that the wild-type LexA contributes to biofilm formation. The decreases was not the result of poor growth of the mutants. These results suggest SOS involvement in hydroxyurea-inducible biofilm formation. In addition, with the paper biofilm assay, it was found that degradation of the biofilm matrix DNA by DNase I appeared to render the biofilms susceptible to the replication inhibitor. The puzzling questions concerning the roles of LexA in DNA release in the biofilm context are discussed.
Assuntos
Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/genética , Resposta SOS em Genética/efeitos dos fármacos , Estresse Fisiológico , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Hidroxiureia/farmacologia , Microscopia Confocal , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Recombinases Rec A/genética , Serina Endopeptidases/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
We demonstrate lasing action with a high spontaneous emission factor and temperature insensitivity in InAs/InGaAs quantum dots (QD) embedded in photonic crystal nanocavities. A quality factor (Q) of over 10,000 was achieved by suppressing the material absorption by QDs uncoupled to the cavity mode. High Q cavities exhibited ultra low threshold lasing with a spontaneous emission factor of 0.7. Less frequent carrier escape from the QDs, which was primarily favored by high potential barrier energy, enabled low threshold lasing up to 90 K.
Assuntos
Arsenicais/química , Gálio/química , Índio/química , Lasers Semicondutores , Nanotecnologia/instrumentação , Pontos Quânticos , Desenho de Equipamento , Análise de Falha de Equipamento , Fótons , Controle de Qualidade , TemperaturaRESUMO
Low-noise millimetre-wave signals are valuable for digital sampling systems, arbitrary waveform generation for ultra-wideband communications, and coherent radar systems. However, the phase noise of widely used conventional signal generators (SGs) will increase as the millimetre-wave frequency increases. Our goal has been to improve commercially available SGs so that they provide a low-phase-noise millimetre-wave signal with assistance from an electro-optics-modulator-based optical frequency comb (EOM-OFC). Here, we show that the phase noise can be greatly reduced by bridging the vast frequency difference between the gigahertz and terahertz ranges with an EOM-OFC. The EOM-OFC serves as a liaison that magnifies the phase noise of the SG. With the EOM-OFC used as a phase noise "booster" for a millimetre-wave signal, the phase noise of widely used SGs can be reduced at an arbitrary frequency f (6 ⦠f ⦠72 GHz).
Assuntos
Óptica e Fotônica/instrumentação , Desenho de Equipamento , Processamento de Sinais Assistido por Computador , Razão Sinal-RuídoRESUMO
Most future information processing techniques using electron spins in non-magnetic semiconductors will require both the manipulation and transfer of spins without their coherence being lost. The spin-orbit effective magnetic field induced by drifting electrons enables us to rotate the electron spins in the absence of an external magnetic field. However, the fluctuations in the effective magnetic field originating from the random scattering of electrons also cause undesirable spin decoherence, which limits the length scale of the spin transport. Here we demonstrate the drift transport of electron spins adjusted to a robust spin structure, namely a persistent spin helix. We find that the persistent spin helix enhances the spatial coherence of drifting spins, resulting in maximized spin decay length near the persistent spin helix condition. Within the enhanced distance of the spin transport, the transport path of electron spins can be modulated by employing time-varying in-plane voltages.
RESUMO
Juvenile hormone (JH) is a key insect growth regulator frequently involved in modulating phenotypically plastic traits such as caste determination in eusocial species, wing polymorphisms in aphids, and mandible size in stag beetles. The jaw morphology of stag beetles is sexually-dimorphic and condition-dependent; males have larger jaws than females and those developing under optimum conditions are larger in overall body size and have disproportionately larger jaws than males raised under poor conditions. We have previously shown that large males have higher JH titers than small males during development, and ectopic application of fenoxycarb (JH analog) to small males can induce mandibular growth similar to that of larger males. What remains unknown is whether JH regulates condition-dependent trait growth in other insects with extreme sexually selected structures. In this study, we tested the hypothesis that JH mediates the condition-dependent expression of the elaborate horns of the Asian rhinoceros beetle, Trypoxylus dichotomus. The sexually dimorphic head horn of this beetle is sensitive to nutritional state during larval development. Like stag beetles, male rhinoceros beetles receiving copious food produce disproportionately large horns for their body size compared with males under restricted diets. We show that JH titers are correlated with body size during the late feeding and early prepupal periods, but this correlation disappears by the late prepupal period, the period of maximum horn growth. While ectopic application of fenoxycarb during the third larval instar significantly delayed pupation, it had no effect on adult horn size relative to body size. Fenoxycarb application to late prepupae also had at most a marginal effect on relative horn size. We discuss our results in context of other endocrine signals of condition-dependent trait exaggeration and suggest that different beetle lineages may have co-opted different physiological signaling mechanisms to achieve heightened nutrient-sensitive weapon growth.
Assuntos
Besouros/anatomia & histologia , Besouros/efeitos dos fármacos , Hormônios Juvenis/farmacologia , Fenilcarbamatos/farmacologia , Animais , Besouros/crescimento & desenvolvimento , Feminino , Hemolinfa/química , Hormônios Juvenis/sangue , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Masculino , Fenótipo , Fenilcarbamatos/sangue , Pupa/efeitos dos fármacos , Pupa/crescimento & desenvolvimento , Caracteres SexuaisRESUMO
A female patient with precursor B-cell acute lymphoblastic leukemia (precursor B-ALL) was analyzed cytogenetically. Karyotyping of the leukemic cells showed a Philadelphia chromosome (Ph1), and also showed a translocation between 2p13 and 14q32, which is thought to be specific for children with B-cell chronic lymphocytic leukemia. DNA analysis with both conventional and pulsed-field gel electrophoresis revealed the rearrangement of the c-abl gene, the BCR gene outside the 5.8 kb breakpoint cluster region (bcr or M-BCR), and the comigration of an abnormal Not I pHabl 5' and 3'-bcr fragment, indicating the presence of BCR/c-abl recombination. The JH gene was rearranged, but the JK gene showed a germline configuration, as with previously reported cases with a t(2;14). This case is the first report of a patient with Ph1-positive precursor B-ALL, in whom a specific translocation t(2;14)(p13;q32) is found simultaneously.
Assuntos
Linfoma de Burkitt/genética , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 2 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Adulto , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Cariotipagem , Translocação GenéticaRESUMO
The genome of mice with the H-2aw18 haplotype has a deletion of approximately 80 kilobases in the H-2 class III region of chromosome 17. Mice that are homozygous for the mutation die soon after birth. A functional form of steroid 21-hydroxylase (21-OHase) is encoded by the deleted DNA fragment, and H-2aw18 homozygotes are deficient in this enzyme. 21-OHase catalyzes the conversion of progesterone to deoxycorticosterone during adrenal steroidogenesis in mice; therefore, H-2aw18 homozygous mice are unable to synthesize corticosteroids. The deleted region also includes the gene for complement component C4, which has a role in the classical pathway of the complement activation cascade. To clarify the cause of the lethality of the mutation, we first administered either an adrenal homogenate or synthetic steroids to newborn mice; as a result, several H-2aw18 homozygotes were rescued. The results demonstrated that the mutant mice die as the result of a defect in adrenal steroidogenesis. The low efficiency of the rescue by treatment of newborns (16.0% by the adrenal homogenate and 14.8% by the synthetic steroids) suggested that mutant mice should be treated prenatally. Moreover, because the 21-OHase gene is expressed before birth, introduction of a gene for 21-OHase should improve the efficiency of rescue. The results of the murine mutation are similar to those of the inherited human disease known as congenital adrenal hyperplasia, which is caused by steroid 21-hydroxylase deficiency. As a model system for treatment of the human disease by genetic therapy, we used transgenic approaches to introduce a recombinant DNA fragment containing the murine genomic gene for 21-OHase into the mutant mice. We produced four lines of transgenic mice, and in all four transgenic lines, the transgene rescued the lethal mutation. The apparent efficiencies of rescue were 80.2%, 80.0%, 68.7%, and 16.7% for the respective lines of transgenic mice. During the course of our experiments, we also found an unexpected property associated with the role of corticosteroids. The H-2aw18 homozygous mice rescued by neonatal treatment survived for a long period without further treatment. This observation indicates that corticosteroids down-stream of 21-OHase in the pathway for adrenal steroidogenesis are not essential for the survival of mice, except during the period immediately after birth.
Assuntos
Corticosteroides/fisiologia , Corticosteroides/uso terapêutico , Hiperplasia Suprarrenal Congênita , Hiperplasia Suprarrenal Congênita/terapia , Animais Recém-Nascidos/fisiologia , Hiperplasia Suprarrenal Congênita/genética , Hiperplasia Suprarrenal Congênita/mortalidade , Animais , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , DNA/análise , DNA/genética , Modelos Animais de Doenças , Feminino , Haplótipos , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Linhagem , Reação em Cadeia da Polimerase , Radioimunoensaio , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/metabolismo , Taxa de SobrevidaRESUMO
The enzyme steroid 21-hydroxylase (21-OHase) plays a key role in adrenal steroidogenesis. Defects in this enzyme are responsible for one of the most common inborn errors of metabolism in humans. Duplicated genes for the enzyme are located in the class III region of the major histocompatibility complex (MHC), HLA. In the mouse, the genes encoding 21-OHase have been mapped to the homologous region of the H-2 complex. We previously described an H-2 recombinant haplotype aw18, in which the gene for the complement component C4 and one of the two genes for 21-OHase in the H-2 class III region have been deleted. We now report that newborn aw18 homozygous mice are deficient in 21-OHase activity, and that homozygosity for the aw18 haplotype directly causes death at the early postnatal stage. Morphological changes in the adrenal glands of newborn aw18 homozygotes are also observed. The aw18 recombinant haplotype is expected to serve as a useful and, thus far, unique experimental system to study adrenal steroidogenesis in vivo and as an animal model for the inherited human disease of congenital adrenal hyperplasia.
Assuntos
Hiperplasia Suprarrenal Congênita , Hiperplasia Suprarrenal Congênita/genética , Camundongos Mutantes/genética , Esteroide Hidroxilases/deficiência , Glândulas Suprarrenais/patologia , Hiperplasia Suprarrenal Congênita/patologia , Animais , Modelos Animais de Doenças , Feminino , Haplótipos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Progesterona/sangueRESUMO
There is compelling evidence that histamine serves as a neurotransmitter in C2, a pair of symmetrical neurons in the cerebral ganglion of Aplysia californica. These cells had previously been shown to contain high concentrations both of histamine and of its biosynthetic enzyme, histidine decarboxylase; in addition, 3H-histamine injected intrasomatically was found to move along C2's axons by fast transport. Furthermore, several actions of C2 on identified follower cells were simulated by the application of histamine. We have now characterized this identified neuron further. C2 converts 3H-histidine to histamine: 16% of the labeled precursor was converted to histamine 1 hour after intrasomatic injection. Synthesis of 3H-histamine is specific, since no conversion occurred after injection of other identified Aplysia neurons that are known to use other neurotransmitter substances. We also examined the fine structure of C2's cell body, axons, and axon terminals within the cerebral ganglion and in the nerves that carry its three peripheral branches, identified after injection of Lucifer Yellow, 3H-histamine, or horseradish peroxidase. Characteristic dense-core vesicles are present in all regions of the neuron, and are labeled after intrasomatic injection of 3H-histamine. These 100-nm vesicles together with 60-nm electron-lucent vesicles fill the varicose extensions of C2's neurites that are widely distributed within the ganglion, but only the smaller vesicles cluster at the membrane specializations presumed to be active zones that make contact with many neurons. The widespread distribution of axon terminals and varicosities is consistent with the idea that C2 is modulatory in function; 3H-histamine is taken up selectively by the cell body and axons of C2 and of several other putative histaminergic neurons in a Na+ -dependent manner. Characterization of these biochemical and morphological features of C2 adds to the large amount of information already available to make this identified cell a standard for identifying other neurons that use histamine as a transmitter.
Assuntos
Aplysia/anatomia & histologia , Histamina/fisiologia , Neurotransmissores/fisiologia , Animais , Aplysia/fisiologia , Autorradiografia , Transporte Axonal , Axônios/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Microscopia Eletrônica , Neurônios/ultraestrutura , Serotonina/metabolismo , Sinapses/ultraestruturaRESUMO
The staging system of limited disease (LD) and extensive disease (ED) is widely used and has been shown to provide useful prognostic information in cases of small cell lung cancer (SCLC). However, accurate examinations are necessary for correct staging. In this report, we evaluated the clinical usefulness of magnetic resonance imaging (MRI) of bone marrow in SCLC. 37 patients with LD by standard staging and 41 with ED were examined with bone marrow MRI. Results of bone marrow MRI did not influence the choice of treatment in patients with LD. For subsequent analysis, patients with LD were divided into two groups: patients in whom bone marrow infiltration was detected with MRI (MRI-positive LD group) and those in whom it was not (MRI-negative LD group). Focal or diffuse metastases to bone marrow were detected with MRI in 46% (36/78) of all patients and 35% (13/37) of LD patients. The response rates to treatment in patients with MRI-positive LD were lower than those in patients with MRI-negative LD (P = 0.006). The survival of patients with MRI-positive LD was worse than that of MRI-negative LD (generalised Wilcoxon test: P = 0.0157), and closer to that of ED. Multivariate analyses using a Cox model that included the result of bone marrow MRI, performance status, chemotherapy regimen, radiotherapy and serum lactose dehydrogenase (LDH) level showed that the result of bone marrow MRI remained a prognostic factor in SCLC patients with limited disease. Bone marrow examination with MRI is useful for better staging of SCLC. According to our analysis of response rates and survival, MRI-positive LD should be considered a type of ED.
Assuntos
Neoplasias da Medula Óssea/secundário , Carcinoma de Células Pequenas/secundário , Neoplasias Pulmonares/patologia , Neoplasias da Medula Óssea/diagnóstico , Neoplasias da Medula Óssea/tratamento farmacológico , Carcinoma de Células Pequenas/diagnóstico , Carcinoma de Células Pequenas/tratamento farmacológico , Humanos , Imageamento por Ressonância Magnética , Estadiamento de Neoplasias/métodos , Prognóstico , Taxa de SobrevidaRESUMO
This article provides a broad overview of our earlier studies on the induction of tumors and congenital anomalies in the progeny of X-irradiated or chemically treated mice and our subsequent (published, hitherto unpublished and on-going) investigations aimed at identifying potential relationships between genetic changes induced in germ cells and the adverse effects manifest as tumors and congenital anomalies using cytogenetic and molecular approaches. The earlier studies document the fact that tumors and congenital anomalies can be induced by irradiation or treatment with certain chemicals such as urethane and that these phenotypes are heritable i.e., transmitted to generations beyond the first generation. These findings support the view that transmissible induced genetic changes are involved. The induced rates of congenital abnormalities and tumors are about two orders of magnitude higher than those recorded in the literature from classical mutation studies with specific locus mutations. The cytogenetic studies addressed the question of whether there were any relationships between induced translocations and induced tumors. The available data permit the inference that gross chromosomal changes may not be involved but do not exclude smaller induced genetic changes that are beyond the resolution of the techniques used in these studies. Other work on possible relationship between visible chromosomal anomalies (in bone marrow preparations) and tumors were likewise negative. However, there were indications that some induced cytogenetic changes might underlie induced congenital anomalies, i.e., trisomies, deletions and inversions were observed in induced and transmissible congenital anomalies (such as dwarfs, tail anomalies). Studies that explored possible relationships between induction of minisatellite mutations at the Pc-3 locus and tumors were negative. However, gene expression analysis of tumor (hepatoma)-susceptible offspring of progeny descended from irradiated male mice showed abnormal expression of many genes. Of these, only very few were oncogenes. This lends some support to our hypothesis that cumulative changes in gene expression of many genes, which perform normal cellular functions, may contribute to the occurrence of tumors in the offspring of irradiated or chemically treated mice.
Assuntos
Anormalidades Induzidas por Medicamentos/genética , Anormalidades Induzidas por Radiação/genética , Cromossomos/genética , Transmissão Vertical de Doenças Infecciosas , Neoplasias Induzidas por Radiação/genética , Síndromes Neoplásicas Hereditárias/genética , 4-Nitroquinolina-1-Óxido/toxicidade , Animais , Carcinógenos/toxicidade , Aberrações Cromossômicas , Cromossomos/efeitos dos fármacos , Cromossomos/efeitos da radiação , Cromossomos/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Genes Letais , Células Germinativas/efeitos dos fármacos , Células Germinativas/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos ICR , Repetições Minissatélites/efeitos dos fármacos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/genética , Neoplasias Induzidas por Radiação/etiologia , Síndromes Neoplásicas Hereditárias/induzido quimicamente , Síndromes Neoplásicas Hereditárias/etiologia , Oncogenes , Dibenzodioxinas Policloradas/toxicidade , Lesões Experimentais por Radiação/genética , Translocação Genética , Uretana/toxicidadeRESUMO
Cultured dorsal root ganglion neurons from newborn rats were mechanically deformed with a fine-tipped glass capillary, and the change in the intracellular Ca2+ concentration ([Ca2+]i) was recorded by Fura-2-based microfluorimetry. The deformation evoked elevation in [Ca2+]i from 18.7 +/- 5.4 nM (mean +/- S.E.M., n = 35) to 137.1 +/- 15.2 nM in some subpopulations of cells, especially those larger than 20 microm in diameter. The largest mechanosensitive cell group was that of cells 20-25 microm in diameter; 56% of the mechanosensitive cells were of this cell size. All of the cells larger than 25 microm in diameter displayed the Ca2+ increase when prodded. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. Verapamil and nickel, blockers of voltage-dependent Ca2+ channels, did not influence the Ca2+ response, whereas gadolinium, a relatively selective blocker of mechanosensitive channels, diminished the response. Na+-free conditions did not influence the response. We concluded that the mechanical stimulation induced a Ca2+ influx in large dorsal root ganglion neurons through mechanosensitive Ca2+-permeable channels.
Assuntos
Canais de Cálcio Tipo N , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Mecanorreceptores/fisiologia , Neurônios Aferentes/metabolismo , Neurônios Aferentes/fisiologia , Animais , Animais Recém-Nascidos/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Citofotometria , Gadolínio/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Neurônios Aferentes/ultraestrutura , Níquel/farmacologia , Estimulação Física , Ratos , Sódio/fisiologia , Verapamil/farmacologiaRESUMO
Thermosensitive cold cells were identified in cultured dorsal root ganglion neurons from newborn rats. The neurons were loaded with a calcium indicator, Fura-PE3, and the change in intracellular Ca2+ concentration ([Ca2+]i) of the neurons was measured with microfluorimetry. Thirteen per cent of the cells responded to the cold stimulation. The diameter of the responder cells was 16.3+/-3.2 microm (mean+/-S.D., n = 25). The lowering of the temperature from 35 degrees C to 20 degrees C increased [Ca2+]i from 59.6+/-10.6 nM to 203.4+/-14.8 nM (n = 25). The [Ca2+]i response was dependent on the intensity of the cold stimulation. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. However, a Na(+)-free condition did not influence the response. We concluded that the cold stimulation opens Ca2(+)-permeable channels in putative cold cells from dorsal root ganglion neurons.
Assuntos
Sinalização do Cálcio/fisiologia , Temperatura Baixa , Gânglios Espinais/citologia , Neurônios Aferentes/fisiologia , Animais , Cálcio/farmacologia , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Masculino , Neurônios Aferentes/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sódio/farmacologiaRESUMO
Pre-treatment of 21-day-old ICR/Jcl mice with 75 micrograms/g of phenobarbital significantly reduced the yields of lung tumors induced by 4-nitroquinoline-1-oxide (4NQO) and furylfuramide. Dimethylbenz[a]anthracene (DMBA)- and urethane-induced lung tumorigenesis was also reduced, but was not significantly different from that in mice receiving only the carcinogen. However, 6 consecutive treatments with phenobarbital (40 micrograms/g) in adult mice greatly reduced urethane-induced lung tumorigenesis. Post-treatment with an equal dose of phenobarbital showed neither diminution nor enhancement of tumorigenesis.
Assuntos
Neoplasias Pulmonares/induzido quimicamente , Fenobarbital/farmacologia , 4-Nitroquinolina-1-Óxido , 9,10-Dimetil-1,2-benzantraceno/farmacologia , Animais , Camundongos , Camundongos Endogâmicos ICR , Neoplasias Experimentais/induzido quimicamente , Fenobarbital/administração & dosagem , Uretana/administração & dosagem , Uretana/farmacologiaRESUMO
The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.
Assuntos
Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Genes Fúngicos , Guanosina Monofosfato/metabolismo , Histidina , Lisina/análogos & derivados , Lisina/análise , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/biossíntese , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Mutação Puntual , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/genéticaRESUMO
Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.