RESUMO
Adherence to medication plays a crucial role in the effective management of chronic diseases. However, patients often miss their scheduled drug administrations, resulting in suboptimal disease control. Therefore, we propose an implantable device enabled with automated and precisely timed drug administration. Our device incorporates a built-in mechanical clock movement to utilize a clockwork mechanism, i.e., a periodic turn of the hour axis, enabling automatic drug infusion at precise 12-h intervals. The actuation principle relies on the sophisticated design of the device, where the rotational movement of the hour axis is converted into potential mechanical energy and is abruptly released at the exact moment for drug administration. The clock movement can be charged either automatically by mechanical agitations or manually by winding the crown, while the device remains implanted, thereby enabling the device to be used permanently without the need for batteries. When tested using metoprolol, an antihypertensive drug, in a spontaneously hypertensive animal model, the implanted device can deliver drug automatically at precise 12-h intervals without the need for further attention, leading to similarly effective blood pressure control and ultimately, prevention of ventricular hypertrophy as compared with scheduled drug administrations. These findings suggest that our device is a promising alternative to conventional methods for complex drug administration.
Assuntos
Fontes de Energia Elétrica , Animais , Humanos , Preparações FarmacêuticasRESUMO
Prompt administration of first-aid drugs can save lives during medical emergencies such as anaphylaxis and hypoglycemia. However, this is often performed by needle self-injection, which is not easy for patients under emergency conditions. Therefore, we propose an implantable device capable of on-demand administration of first-aid drugs (i.e., the implantable device with a magnetically rotating disk [iMRD]), such as epinephrine and glucagon, via a noninvasive simple application of the magnet from the outside skin (i.e., the external magnet). The iMRD contained a disk embedded with a magnet, as well as multiple drug reservoirs that were sealed with a membrane, which was designed to rotate at a precise angle only when the external magnet was applied. During this rotation, the membrane on a designated single-drug reservoir was aligned and torn to expose the drug to the outside. When implanted in living animals, the iMRD, actuated by an external magnet, delivers epinephrine and glucagon, similar to conventional subcutaneous needle injections.
RESUMO
BACKGROUND: Recently developed imaging techniques, such as cone beam computed tomography (CBCT) and CAD/CAM technology, have facilitated reliable implant planning and implant surgical guide production by 3D printing. This study compared the accuracy of implant-guided surgery using the R2GATE® program with CBCT before and after surgery. PATIENTS AND METHODS: The study included patients who visited the Department of Oral and Maxillofacial Surgery at Chonnam National University Hospital from September 2021 to March 2022. Twenty-four implants were placed in eleven patients. Using R2GATE® Windows (Megagen implant, Daegu, Korea) software, implant placement was planned. The difference was measured by the CBCT before and after surgery. The cervical and apical distance and angular deviation of the implants were measured. Statistical analysis was performed using an independent t-test, Pearson correlation, and multiple regression analyses. RESULTS: The three-dimensional linear distance difference between the planned implant and the placed implant was 0.97 ± 0.37 mm at the cervical and 1.13 ± 0.36 mm at the apical. The difference in angle deviation between the planned implant and the placed implant was 3.42 ± 2.12°. Among the variables affecting the accuracy of implant placement, a statistically significant difference was found when using a tissue-supported implant guide, implant diameter and implant length. CONCLUSION: Based on these results, using the R2GATE® program is useful to use an implant digital surgical guide, and it will be used in various clinic.
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The keratinocytes in UV-irradiated skin produce and secrete α-melanocyte-stimulating hormone. α-Melanocyte-stimulating hormone upregulates the expression of MITF in melanocytes through the cAMPâprotein kinase AâCREB signaling pathway. Thereafter, MITF induces the expression of melanogenic genes, including the tyrosinase gene TYR and TYRP-1 and TYRP-2 genes, which leads to the synthesis and accumulation of melanin. In this study, we examined whether MITF basic region-derived tripeptides can bind to the DNA-binding domain of MITF and inhibit MITF-induced melanogenesis through the inhibition of MITFâDNA binding. MITF-KGR, a representative MITF-derived tripeptide, suppressed the transcriptional activity of MITF by disrupting its binding to the promoter region of the target genes, which resulted in the inhibition of skin epidermis thickness and melanin synthesis in vivo and in vitro. Our results indicate that MITF-KGR exerts an inhibitory effect on melanogenesis by targeting MITF.
Assuntos
Fator de Transcrição Associado à Microftalmia/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Regiões Promotoras Genéticas , Animais , Linhagem Celular Tumoral , DNA/metabolismo , Oxirredutases Intramoleculares/genética , Melaninas/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/genética , Oxirredutases/genética , Raios Ultravioleta , alfa-MSH/antagonistas & inibidoresRESUMO
Atopic dermatitis (AD) is one of the most common skin diseases with inflammation, chronic relapses, and intense pruritus. Its pathogenesis includes genetic susceptibility, an abnormal epidermal lipid barrier, and an increased production of IgE due to immune dysregulation. Recently, AD has been reported to be associated with intestinal inflammation and dysbiosis in human and murine models. Various probiotics are being used to control intestinal dysbiosis and inflammatory reactions. However, it is difficult to predict or determine the therapeutic effects of the probiotics, since it is rare for clinicians to use the probiotics alone to treat AD. It is also difficult to check whether the intestinal inflammation in patients with AD has improved since probiotic treatment. The aim of the present study was to determine whether mice with induced atopic dermatitis had any changes in fecal calprotectin, an indicator of intestinal inflammation, after probiotic administration. Our results showed that the fecal calprotectin levels in mice with induced dermatitis decreased significantly after the administration of probiotics. In addition, epidermal skin lesions were attenuated and inflammatory-related cytokines were downregulated after the administration of probiotics in mice with induced dermatitis. These results suggest that changes in fecal calprotectin levels could be used to assess the effectiveness of a probiotic strain as an adjuvant treatment for AD.
Assuntos
Dermatite Atópica/terapia , Inflamação/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Probióticos/farmacologia , Administração Oral , Animais , Citocinas/metabolismo , Dermatite Atópica/microbiologia , Modelos Animais de Doenças , Fezes/química , Feminino , Microbioma Gastrointestinal , Camundongos , Reação em Cadeia da Polimerase , Prurido/metabolismo , Recidiva , Pele/metabolismoRESUMO
In canonical pathway, Wnt3A has been known to stabilize ß-catenin through the dissociation between ß-catenin and glycogen synthase kinase-3ß (GSK-3ß) that suppresses the phosphorylation and degradation of ß-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3ß and accumulation of ß-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3ß and accumulation of ß-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3ß phosphorylation and ß-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3ß in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3ß phosphorylation and ß-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Wnt3A/farmacologia , beta Catenina/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Piridinas/farmacologia , Células RAW 264.7 , Proteínas Recombinantes de Fusão/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Cryparin is an abundant cell-wall-associated hydrophobin of Cryphonectria parasitica. Although cryparin is encoded as a single copy gene, it is the most abundant protein produced by this fungus when grown in liquid culture. Studies to characterize the transcriptional regulatory element(s) found that the fragment between nt -188 and the start codon was the minimal but sufficient promoter element for expression of the cryparin gene. To explore the possibility of using this small fragment as a minimal core promoter, three different chimeric reporter genes were constructed using a bacterial hygromycin B resistance gene (hph), an inducible laccase of C. parasitica, and glucose oxidase of Aspergillus niger to examine the promoter properties of the fragment. When using C. parasitica as an expression host, the 188-bp fragment functioned as a promoter for the expression of all three reporter genes. Moreover, a high level of expression was further confirmed by measuring the relative amount of transcripts of hph and an internal control gene, glyceraldehyde-3-phosphate dehydrogenase, using quantitative real-time polymerase chain reaction. The 188-bp fragment also showed promoter activity in other fungi, Gibberella zeae, A. niger, and Aspergillus nidulans, which suggests that this fragment can be applied as a strong core promoter for heterologous gene expression in various fungi.
Assuntos
Proteínas Fúngicas/genética , Fungos/genética , Expressão Gênica , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Sequência de Bases , Fungos/metabolismo , Genes Reporter , Dados de Sequência Molecular , Deleção de SequênciaRESUMO
Cryparin, encoded as a single copy gene (Crp) of the chestnut blight fungus Cryphonectria parasitica, is the most abundant protein produced by this fungus. However, its accumulation is decreased remarkably in C. parastica strains containing the double-stranded (ds) RNA virus Cryphonectria hypovirus 1. To characterize the transcriptional regulatory element(s) for strong expression and viral regulation, promoter analysis was conducted. Serial deletion of the Crp promoter region resulted in a step-wise decrease in promoter activity, indicating a localized distribution of genetic elements in the cryparin promoter. Promoter analysis indicated two positive and a repressive cis-acting elements. Among them, the promoter region between nt -1,282 and -907 appeared to be necessary for hypoviral-mediated down-regulation. An electrophoretic mobility shift assay (EMSA) on the corresponding promoter region (-1,282/-907) indicated two regions at (-1257/-1158) and (-1107/-1008) with the characteristic AGGAGGA-N42-GAGAGGA and its inverted repeat TCCTCTC-N54-TCCTCCT, respectively, appeared to be specific binding sites for cellular factors.
Assuntos
Ascomicetos/genética , Ascomicetos/virologia , Membrana Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Vírus de RNA/fisiologia , Ascomicetos/citologia , Pareamento de Bases , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Transformação GenéticaRESUMO
We analysed 676 isolates from 33 Korean Cryphonectria parasitica subpopulations in Korea for dsRNA incidence and diversity. dsRNA was detected in 84 isolates. Although the dsRNA banding patterns varied in several minor bands, infected isolates could be categorized into two groups. The most common banding pattern occurred in 77 isolates and contained a 12.7-kb band indicative of Cryphonectria hypovirus 1 (CHV1), and several accompanying minor bands with sizes ranging from 0.9-5kb. Northern blot analysis revealed that all 12.7-kb fragments in the dsRNA-containing isolates hybridized to probes corresponding to open reading frames (ORFs) A and B from the reference CHV1 strain (GenBank accession no. M57938). In addition, the sequence of a 1.4-kb cDNA fragment from a representative isolate of the most common group showed 99% sequence similarity to ORF A of CHV1. However, the other group of seven isolates had distinctive bands of 3.5 and 3.3kb, but not the 12.7-kb band. Sequence comparison showed that cloned fragments of these dsRNAs were similar to those of the coat protein and RNA-dependent RNA polymerase genes of chrysovirus, which indicates the occurrence of chrysovirus in the Korean population. Fungal strain identity was assessed via RFLP analysis of the ITS regions. Among the 84 tested isolates, six had different ITS-RFLP patterns (RFLP-II) from that (RFLP-I) of C. parasitica, and are believed to be C. nitschkei, a sympatric species reported on chestnut trees in Japan. The chrysovirus and CHV1 were detected in strains showing both RFLP patterns. However, the chrysovirus was more frequent in the RFLP-II group.
Assuntos
Ascomicetos/virologia , DNA Viral/isolamento & purificação , Fagaceae/microbiologia , Doenças das Plantas/microbiologia , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/isolamento & purificação , Sequência de Aminoácidos , Ascomicetos/genética , Ascomicetos/isolamento & purificação , DNA Viral/genética , Coreia (Geográfico) , Dados de Sequência Molecular , Fases de Leitura Aberta , Vírus de RNA/química , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , Alinhamento de SequênciaRESUMO
Lichen nitidus (LN) is an uncommon chronic inflammatory skin disease composed of numerous, tiny, shiny, flesh-colored papules that are predominantly observed on the chest, abdomen, glans penis and upper extremities. The distribution of LN is most often localized, but in some cases it can become generalized. Because LN tends to be asymptomatic and presents spontaneous resolution within several years, it usually does not require treatment except in symptomatic, persistent and generalized cases. We describe a 28-yr-old man and a 7-yr-old boy with generalized LN where both cases improved with narrow-band ultraviolet B (NB-UVB) phototherapy plus topical steroid ointment. Both patients noted improvement within the first three treatments and showed almost complete resolution after 18 and 20 treatments, respectively. NB-UVB phototherapy may be an effective alternative therapy for the treatment of generalized LN, even for those patients in their childhood.
Assuntos
Líquen Nítido/radioterapia , Terapia Ultravioleta , Adulto , Criança , Humanos , Líquen Nítido/patologia , Masculino , Terapia PUVARESUMO
Yeast cell walls are critical for maintaining cell integrity, particularly in the face of challenges such as growth in mammalian hosts. The pathogenic fungus Cryptococcus neoformans additionally anchors its polysaccharide capsule to the cell surface via alpha(1-3) glucan in the wall. Cryptococcal cells disrupted in their alpha glucan synthase gene were sensitive to stresses, including temperature, and showed difficulty dividing. These cells lacked surface capsule, although they continued to shed capsule material into the environment. Electron microscopy showed that the alpha glucan that is usually localized to the outer portion of the cell wall was absent, the outer region of the wall was highly disorganized, and the inner region was hypertrophic. Analysis of cell wall composition demonstrated complete loss of alpha glucan accompanied by a compensatory increase in chitin/chitosan and a redistribution of beta glucan between cell wall fractions. The mutants were unable to grow ina mouse model of infection, but caused death in nematodes. These studies integrate morphological and biochemical investigations of the role of alpha glucan in the cryptococcal cell wall.
Assuntos
Parede Celular/química , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Glucanos/fisiologia , Animais , Caenorhabditis elegans/microbiologia , Parede Celular/genética , Parede Celular/ultraestrutura , Quitina/análise , Quitosana/análise , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Modelos Animais de Doenças , Deleção de Genes , Glucosiltransferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mutagênese Insercional , Virulência/genética , beta-Glucanas/análiseRESUMO
Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica is known to downregulate the fungal laccase1 (lac1), the modulation of which is tightly governed by the inositol triphosphate (IP(3)) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), to investigate the regulation of lac1 expression and to better characterize fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that the protein product contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd beta-strand and the alpha-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a delta-type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. Accordingly, reduced virulence of the cplc1-null mutant as compared to the wild-type was observed, which can be ascribed to the growth defect. However, other PLC-associated characteristics including temperature sensitivity and osmosensitivity did not differ from the wild-type strain. Northern blot analysis revealed no accumulation of the lac1 gene transcript due to the disruption of the cplc1 gene. Functional complementation of the cplc1-null mutant with the PLC1 gene from Saccharomyces cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for normal mycelial growth rate and colony morphology, and that it regulates the lac1 expression, which is also modulated by the hypovirus. Although several PLC genes have been identified in various simple eukaryotic organisms, the deletion analysis of the cplc1 gene in this study appears to be the first report on the functional analysis of PLC in filamentous fungi.
Assuntos
Ascomicetos/enzimologia , Regulação Fúngica da Expressão Gênica , Lacase/biossíntese , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Ascomicetos/citologia , Ascomicetos/genética , Ascomicetos/fisiologia , Northern Blotting , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Teste de Complementação Genética , Crescimento/genética , Dados de Sequência Molecular , Morfogênese/genética , Mutagênese Insercional , Micélio/genética , Pigmentação/genética , Casca de Planta/microbiologia , Doenças das Plantas/microbiologia , Estrutura Terciária de Proteína , RNA Fúngico/análise , RNA Mensageiro/análise , Alinhamento de Sequência , Análise de Sequência de DNA , Virulência/genéticaRESUMO
The Cryphonectria parasitica gene cpmk2, which encodes a mitogen-activated protein kinase belonging to the yeast extracellular signalling-regulated kinase (YERK1) subfamily, was isolated and its biological function was examined. Disruption of cpmk2 resulted in impaired pigmentation and abolished conidiation. Growth defects were observed in the cpmk2 mutant grown on solid plates, but growth of the mutant appeared normal in liquid media, including EP complete and PD broth, suggesting that the cpmk2 gene is involved in sensing and responding to growth conditions. The mutant's production of laccase, as measured by the size of the coloured area produced on tannic-acid-supplemented plates, was significantly reduced compared with the wild-type, but the intensity of the coloured area was unchanged, suggesting that the reduced laccase activity was owing to reduced growth on solid media rather than transcriptional downregulation. A dramatic reduction observed in the canker area produced by the cpmk2 mutant compared with the wild-type, even more severe than that of a hypovirulent strain, can also be ascribed to defective growth on solid surfaces rather than to impairments in a virulence factor(s). Downregulation of the pheromone gene Mf2/1 was also observed in the mutant, indicating a possible explanation for the regulation of the pheromone precursor gene in filamentous fungi and suggesting the presence of the yeast-like pheromone-responsive pathway in C. parasitica. Immunoblot analyses revealed that the phosphorylation level of CpMK2 increased in both virus-free and virus-containing strains in liquid cultures of up to 5 days old and decreased in older cultures. Moreover, the CpMK2 phosphorylation level increased in both strains after transfer from liquid to solid medium. However, levels of phosphorylated CpMK2 were similar in the two strains, suggesting that CpMK2, unlike CpMK1, is not under the direct control of a hypovirus.
Assuntos
Ascomicetos/enzimologia , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno , Doenças das Plantas/microbiologia , Árvores/microbiologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Ascomicetos/virologia , Meios de Cultura , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Immunoblotting , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/isolamento & purificação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Feromônios/genética , Feromônios/metabolismo , Fosforilação , VirulênciaRESUMO
The cppk1 gene encodes a Ser/Thr protein kinase of Cryphonectria parasitica and is transcriptionally up-regulated by the presence of hypovirus CHV1-EP713. A cppk1-null mutant was constructed to determine the function of cppk1. The cppk1-null mutant was initially isolated as a heterokaryotic form containing both wild-type and cppk1-deleted nuclei. The pure cppk1-null homokaryon was obtained by the single spore isolation of the heterokaryon. While the parental heterokaryon appeared normal, the pure cppk1-null mutant exhibited dramatic changes in colony morphology. It showed characteristics of microcolonial growth. The functional complementation, restoration of filamentous growth, of the cppk1-null mutant using a wild-type cppk1 gene indicated that the phenotypic changes were due to the disruption of cppk1. Neither sporulation nor hyphal differentiation into feeding hyphae, a mycelial mat, or aerial hyphae was observed in the cppk1-null mutant. Instead of bright yellow, dark brown pigmentation appeared as the culture grew. Hyphae were shortened and hyperbranched with globose to bulbose cells. Electron microscopy revealed the presence of intrahyphal hyphae, the most striking ultrastructural change. Subtle changes in the expression of CpPK1 resulted in abnormalities in colony morphology and pigmentation, which indicated that cppk1 is important for coordinating growth with development and maintaining cell wall integrity.
Assuntos
Ascomicetos/enzimologia , Ascomicetos/crescimento & desenvolvimento , Deleção de Genes , Genes Fúngicos , Proteínas Serina-Treonina Quinases/genética , Ascomicetos/citologia , Ascomicetos/genética , Parede Celular/genética , Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Hifas/citologia , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/ultraestrutura , Pigmentação/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Fúngico/análise , RNA Mensageiro/análise , Esporos Fúngicos/citologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
We examined the biological function of cpmk1, which encodes a MAPK of Cryphonectria parasitica, and its regulation by mycovirus. Sequence comparisons revealed that cpmk1 had highest homology with osm1, a hog1-homologue from Magnaporthe grisea. A growth defect was observed in the cpmk1-null mutant under hyperosmotic conditions, indicating that cpmk1 functionally belongs to a hog1 subfamily. Immunoblot analyses indicated that the CpMK1 pathway was affected specifically in hyperosmotic conditions by the hypovirus CHV1-EP713. Moreover, the virus-infected hypovirulent UEP1 strain also exhibited severe osmosensitivity compared to the virus-free isogenic strain EP155/2, thus providing additional evidence for viral regulation of cpmk1 in response to a hypertonic stress. Besides osmosensitivity, disruption of cpmk1 resulted in several, but not all, hypovirulence-associated changes, such as reduced pigmentation, conidiation, laccase production and cryparin expression. However, the cpmk1-null mutant exhibited an increased accumulation of pheromone gene transcripts. Virulence assays of the cpmk1-null mutant revealed reduced canker area, but not as severe as that of UEP1. These results suggest that mycoviruses modulate the MAPK and thereby provoke the aberrant expression of target genes, some of which are likely to be implicated in viral symptom development.
Assuntos
Ascomicetos/metabolismo , Ascomicetos/virologia , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Vírus de RNA/fisiologia , Sequência de Aminoácidos , Ascomicetos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Soluções Hipertônicas , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Fenótipo , Fosforilação , Filogenia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Virulência/fisiologiaRESUMO
The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coli-derived CpPK1 revealed CpPK1-specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell-free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus-free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.