RESUMO
The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.
Assuntos
Acroleína/análogos & derivados , Lignina/metabolismo , Oxigenases de Função Mista/metabolismo , Oryza/genética , Acroleína/metabolismo , Biomassa , Sistemas CRISPR-Cas , Parede Celular/metabolismo , Ácidos Cumáricos , Mutação com Perda de Função , Oxigenases de Função Mista/genética , Oryza/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Propionatos/metabolismoRESUMO
Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.
Assuntos
Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , Oryza/genética , Propionatos/metabolismo , Fatores de Transcrição/metabolismo , Biomassa , Sistemas CRISPR-Cas , Parede Celular/química , Parede Celular/metabolismo , Ácidos Cumáricos , Edição de Genes , Redes Reguladoras de Genes , Lignina/química , Mutação com Perda de Função , Oryza/química , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Striga species are parasitic weeds that seriously constrain the productivity of food staples, including cereals and legumes, in Sub-Saharan Africa and Asia. In eastern and central Africa, Striga spp. infest as much as 40 million hectares of smallholder farmland causing total crop failure during severe infestation. As the molecular mechanisms underlying resistance are yet to be elucidated, we undertook a comparative metabolome study using the Striga-resistant rice (Oryza sativa) cultivar 'Nipponbare' and the susceptible cultivar 'Koshihikari'. We found that a number of metabolites accumulated preferentially in the Striga-resistant cultivar upon Striga hermonthica infection. Most apparent was increased deposition of lignin, a phenylpropanoid polymer mainly composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatic units, around the site of interaction in Nipponbare. The increased deposition of lignin was accompanied by induction of the expression of corresponding enzyme-encoding genes in the phenylpropanoid pathway. In addition, perturbing normal lignin composition by knocking down or overexpressing the genes that regulate lignin composition, i.e. p-COUMARATE 3-HYDROXYLASE or FERULATE 5-HYDROXYLASE, enhanced susceptibility of Nipponbare to S hermonthica infection. These results demonstrate that enhanced lignin deposition and maintenance of the structural integrity of lignin polymers deposited at the infection site are crucial for postattachment resistance against S hermonthica.
Assuntos
Interações Hospedeiro-Parasita/genética , Lignina/química , Oryza/genética , Striga/fisiologia , Lignina/genética , Oryza/parasitologia , Doenças das Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/parasitologiaRESUMO
p-Coumaroyl ester 3-hydroxylase (C3'H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3'H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3'H deficiency on the structure and properties of grass cell walls. C3'H-knockdown lines generated via RNA interference (RNAi)-mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3'H-knockout rice mutants generated via CRISPR/Cas9-mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3'H-knockdown RNAi lines revealed that their lignins were largely enriched in p-hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non-acylated lignin units, with grass-specific γ-p-coumaroylated lignin units remaining apparently unchanged. Suppression of C3'H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross-linking ferulates. Collectively, our data demonstrate that C3'H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross-linking. We also demonstrated that C3'H-suppressed rice displays enhanced biomass saccharification.
RESUMO
In rice (Oryza sativa), OsF2H and OsFNSII direct flavanones to independent pathways that form soluble flavone C-glycosides and tricin-type metabolites (both soluble and lignin-bound), respectively. Production of soluble tricin metabolites requires CYP75B4 as a chrysoeriol 5'-hydroxylase. Meanwhile, the close homologue CYP75B3 is a canonical flavonoid 3'-hydroxylase (F3'H). However, their precise roles in the biosynthesis of soluble flavone C-glycosides and tricin-lignins in cell walls remain unknown. We examined CYP75B3 and CYP75B4 expression in vegetative tissues, analyzed extractable flavonoid profiles, cell wall structure and digestibility of their mutants, and investigated catalytic activities of CYP75B4 orthologues in grasses. CYP75B3 and CYP75B4 showed co-expression patterns with OsF2H and OsFNSII, respectively. CYP75B3 is the sole F3'H in flavone C-glycosides biosynthesis, whereas CYP75B4 alone provides sufficient 3',5'-hydroxylation for tricin-lignin deposition. CYP75B4 mutation results in production of apigenin-incorporated lignin and enhancement of cell wall digestibility. Moreover, tricin pathway-specific 3',5'-hydroxylation activities are conserved in sorghum CYP75B97 and switchgrass CYP75B11. CYP75B3 and CYP75B4 represent two different pathway-specific enzymes recruited together with OsF2H and OsFNSII, respectively. Interestingly, the OsF2H-CYP75B3 and OsFNSII-CYP75B4 pairs appear to be conserved in grasses. Finally, manipulation of tricin biosynthesis through CYP75B4 orthologues can be a promising strategy to improve digestibility of grass biomass for biofuel and biomaterial production.
Assuntos
Vias Biossintéticas , Flavonas/metabolismo , Flavonoides/metabolismo , Metaboloma , Oxigenases de Função Mista/metabolismo , Poaceae/metabolismo , Metabolismo dos Carboidratos , Parede Celular/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonas/química , Flavonoides/química , Regulação da Expressão Gênica de Plantas , Glicosídeos/metabolismo , Lignina/metabolismo , Espectroscopia de Ressonância Magnética , Mutação/genética , Oryza/metabolismo , Panicum/metabolismo , Solubilidade , Sorghum/metabolismoRESUMO
Parasitic plants in the family Orobanchaceae are destructive weeds of agriculture worldwide. The haustorium, an essential parasitic organ used by these plants to penetrate host tissues, is induced by host-derived phenolic compounds called haustorium-inducing factors (HIFs). The origin of HIFs remains unknown, although the structures of lignin monomers resemble that of HIFs. Lignin is a natural phenylpropanoid polymer, commonly found in secondary cell walls of vascular plants. We therefore investigated the possibility that HIFs are derived from host lignin. Various lignin-related phenolics, quinones and lignin polymers, together with nonhost and host plants that have different lignin compositions, were tested for their haustorium-inducing activity in two Orobanchaceae species, a facultative parasite, Phtheirospermum japonicum, and an obligate parasite, Striga hermonthica. Lignin-related compounds induced haustoria in P. japonicum and S. hermonthica with different specificities. High concentrations of lignin polymers induced haustorium formation. Treatment with laccase, a lignin degradation enzyme, promoted haustorium formation at low concentrations. The distinct lignin compositions of the host and nonhost plants affected haustorium induction, correlating with the response of the different parasitic plants to specific types of lignin-related compounds. Our study provides valuable insights into the important roles of lignin biosynthesis and degradation in the production of HIFs.
Assuntos
Interações Hospedeiro-Parasita , Lignina/metabolismo , Orobanchaceae/anatomia & histologia , Striga/anatomia & histologia , Antocianinas/metabolismo , Arabidopsis/parasitologia , Vias Biossintéticas , Oryza/parasitologia , Plantas Geneticamente Modificadas , Quinonas/metabolismoRESUMO
Lignin, a ubiquitous phenylpropanoid polymer in vascular plant cell walls, is derived primarily from oxidative couplings of monolignols (p-hydroxycinnamyl alcohols). It was discovered recently that a wide range of grasses, including cereals, utilize a member of the flavonoids, tricin (3',5'-dimethoxyflavone), as a natural comonomer with monolignols for cell wall lignification. Previously, we established that cytochrome P450 93G1 is a flavone synthase II (OsFNSII) indispensable for the biosynthesis of soluble tricin-derived metabolites in rice (Oryza sativa). Here, our tricin-deficient fnsII mutant was analyzed further with an emphasis on its cell wall structure and properties. The mutant is similar in growth to wild-type control plants with normal vascular morphology. Chemical and nuclear magnetic resonance structural analyses demonstrated that the mutant lignin is completely devoid of tricin, indicating that FNSII activity is essential for the deposition of tricin-bound lignin in rice cell walls. The mutant also showed substantially reduced lignin content with decreased syringyl/guaiacyl lignin unit composition. Interestingly, the loss of tricin in the mutant lignin appears to be partially compensated by incorporating naringenin, which is a preferred substrate of OsFNSII. The fnsII mutant was further revealed to have enhanced enzymatic saccharification efficiency, suggesting that the cell wall recalcitrance of grass biomass may be reduced through the manipulation of the flavonoid monomer supply for lignification.
Assuntos
Biomassa , Lignina/metabolismo , Oxigenases de Função Mista/metabolismo , Oryza/enzimologia , Vias Biossintéticas/genética , Parede Celular/metabolismo , Fertilidade/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Espectroscopia de Ressonância Magnética , Mutação/genética , Oryza/genética , FenótipoRESUMO
MAIN CONCLUSION: Regulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.
Assuntos
Carboxiliases/metabolismo , Lignina/metabolismo , Oryza/enzimologia , Acroleína/análogos & derivados , Acroleína/química , Acroleína/metabolismo , Biocombustíveis , Biomassa , Vias Biossintéticas , Carboxiliases/genética , Parede Celular/metabolismo , Regulação para Baixo , Engenharia Genética , Lignina/química , Oryza/citologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Filogenia , Folhas de Planta/anatomia & histologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regulação para CimaRESUMO
While metallic biomaterials have led to an improvement in the quality of life, metal allergies, especially to palladium (Pd), has caused a recent increase in allergic patients. Metal allergy is known to be a T cell-mediated delayed-type hypersensitivity (DTH); however, the pathogenic T cell subsets and the specific T cell receptor (TCR) have not been identified. Therefore, we attempted to identify the pathogenic T cells responsible for Pd allergy. We found that activating CD8⺠T cells significantly increased and that the TRAV (TCRα variable) 7-2*02 chain skewed in Pd allergic mice. Furthermore, adoptive transfer experiments revealed that in vitro-cultured Pd-stimulated antigen presenting cells (APCs) function as memory APCs with recipient mice developing Pd allergy and that the frequency of TRAV7-2*02 increases the same as conventional Pd allergic mice. In contrast, neither proliferation of CD8⺠T cells nor increasing of TRAV7-2*02 was observed in major histocompatibility complex I (MHC I)-deficient Pd-APCs transferred to mice. Taken together, we revealed that TRAV7-2*02-expressing CD8⺠T cells are the pathogenic T cells for the development of Pd allergy. We also identified the CDR3 consensus motif of pathogenic TCRs as CAAXSGSWQLIF in TRAV7-2*02/TRAJ (TCRα junction)22*01 positive cells. These results suggest that the specific TCRs represent novel targets for the development of diagnostics and treatments for metal allergy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hipersensibilidade/imunologia , Paládio/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/transplante , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Hipersensibilidade/genética , Hipersensibilidade/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
BACKGROUND & AIMS: Fetuin-A (α2HS-glycoprotein), a liver secretory glycoprotein, is known as a transforming growth factor (TGF)-ß1 signalling inhibitor. Serum fetuin-A concentration is associated with nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease. However, the usefulness of serum fetuin-A as a predictive fibrosis biomarker in NAFLD patients remains unclear. In this study, we investigated the relationship between circulating fetuin-A levels and fibrosis-related markers [platelet count, NAFLD fibrosis score and carotid intima media thickness (IMT)] in subjects with NAFLD. METHODS: A total of 295 subjects (male, 164; female, 131) who received medical health check-ups were enrolled in this study. NAFLD was diagnosed using abdominal ultrasonography. Serum fetuin-A was measured by ELISA. IMT was assessed using a high-resolution ultrasound scanner. Using recombinant human fetuin-A, we investigated the effects of fetuin-A on hepatic stellate cells, which play a pivotal role in the process of hepatic fibrosis. RESULTS: Serum fetuin-A concentration was significantly correlated with platelet count (R = 0.19, P < 0.01), NAFLD fibrosis score (R = -0.25, P < 0.01) and mean IMT (R = -0.22, P < 0.01). Multivariate analyses revealed that the fetuin-A concentration is a significant and independent determinant of platelet count, NAFLD fibrosis score and mean IMT. Recombinant fetuin-A suppressed TGF-ß1 signalling and fibrosis-related gene expression and increased the expression of TGF-ß1 pseudoreceptor bone morphogenic protein and activin membrane-bound inhibitor (BAMBI). CONCLUSIONS: Serum fetuin-A level is associated with liver/vessel fibrosis-related markers in NAFLD patients. Circulating fetuin-A could be a useful serum biomarker for predicting liver and vascular fibrosis progression in NAFLD patients.
Assuntos
Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/sangue , alfa-2-Glicoproteína-HS/metabolismo , Idoso , Biomarcadores/sangue , Espessura Intima-Media Carotídea , Linhagem Celular , Feminino , Fibrose , Células Estreladas do Fígado/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Contagem de Plaquetas , Fator de Crescimento Transformador beta1RESUMO
BACKGROUND: Fucosylation is one of the most important glycosylation events involved in cancer and inflammation. We previously developed a lectin antibody ELISA kit to measure fucosylated haptoglobin (Fuc-Hpt), which we identified as a novel cancer biomarker. In this study, we investigated Fuc-Hpt as a biomarker in chronic liver diseases, especially in hepatocellular carcinoma (HCC). METHODS: We measured serum Fuc-Hpt levels using our ELISA kit in 318 patients with chronic liver diseases, including 145 chronic hepatitis (CH) patients, 81 liver cirrhosis (LC) patients, and 92 HCC patients. During a long-term follow-up period of 7 years (1996-2003), Fuc-Hpt levels were measured at three different time points in 19 HCC patients. Serum Fuc-Hpt levels were also examined with a short-term follow-up period of 3 years (2009-2012) in 13 HCC patients. RESULTS: Fuc-Hpt levels increased with liver disease progression. Patients with LC and HCC showed significantly increased Fuc-Hpt levels in comparison to CH patients or healthy volunteers. Fuc-Hpt levels tended to be higher in HCC patients than in LC patients. Fuc-Hpt was better than α-fetoprotein (AFP) and AFP-L3 for predicting HCC [diagnosed by computed tomography (CT) or ultrasound] in LC patients with long-term follow-up. More than 80% of LC patients with long-term follow-up showed increased Fuc-Hpt during hepatocarcinogenesis, and 38% of early-stage HCC patients with short-term follow-up showed a gradual increase in Fuc-Hpt before imaging diagnosis. CONCLUSIONS: These results suggest that Fuc-Hpt is a novel and potentially useful biomarker for predicting liver disease progression and HCC development.
Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/sangue , Fucose/metabolismo , Haptoglobinas/metabolismo , Neoplasias Hepáticas/sangue , Progressão da Doença , Detecção Precoce de Câncer , Humanos , Neoplasias Hepáticas/diagnósticoRESUMO
In patients with unilateral mandibular condyle hyperplasia, whether to perform condylectomy and orthognathic surgical procedures at the same time or orthognathic surgery in two stages for remains controversial. Reported here is a case of facial asymmetry with mandibular condyle hyperplasia, for which condylectomy and orthognathic surgery procedures were performed at the same time. A 28-year-old woman was presented to our department with chief complaints of left deviation of the mandible and right temporomandibular joint (TMJ) noise. Findings obtained in several imaging examinations led to a diagnosis of facial asymmetry associated with right mandibular condyle hyperplasia. Following 3 months of preoperative orthodontic treatment, in October 2018 under general anesthesia the patient underwent a right mandibular condylectomy, Le Fort I osteotomy, right mandibular sagittal split ramus osteotomy, and left mandibular inverted L ramus osteotomy. In examinations up to 3 years after surgery, good results were noted. For this case of severe facial asymmetry with mandibular condyle hyperplasia, early surgery and condylectomy were performed simultaneously to significantly shorten the total treatment time. The effectiveness of a surgery-early approach was confirmed by no postoperative findings indicating abnormalities in the TMJ or retroversion.
RESUMO
A 36-year-old female was pointed out to have liver enzyme elevation by routine health checkup. Subsequent contrast-enhanced CT scan identified gigantic uterine fibroids and retroperitoneal tumor. She was referred to the gynecologist at JA Toride Medical Center and planned to undergo a uterus enucleation and biopsy of the retroperitoneal tumor. The surgery was conducted without any troubles. After the surgery, the patient presented polyuria with urine volume 10-20 L a day and developed hypovolemic shock. Laboratory test revealed hypotonic urine and hypernatremia. Arginine vasopressin (AVP) loading test suggested shortage of endogenous vasopressin. Since the subcutaneous administration of AVP was not sufficient to control the urine volume, continuous intravenous infusion of AVP was initiated. After achieving hemodynamic stability, the treatment was switched to oral desmopressin. MRI finding indicated attenuation of high signal in posterior pituitary in T1 weighted image while neither enlargement of pituitary nor thickening of pituitary stalk was indicated by enhanced MRI. Hypertonic salt solution test indicated no responsive elevation of AVP, confirming the diagnosis of central diabetes insipidus (CDI). Her anterior pituitary function was preserved. Only anti-rabphilin-3A antibody was found positive in the serum of the patient, while other secondary causes for CDI were denied serologically and radiologically. Hence, lymphocytic infundibuloneurohypophysitisã(LINH) was suspected as the final diagnosis. Hormonal replacement therapy by nasal desmopressin was continued and the patient managed to control her urine volume. In cases of CDI considered idiopathic with conventional examinations, anti-rabphilin-3A antibody may be a clue for determining the cause as LINH.
Assuntos
Diabetes Insípido Neurogênico , Diabetes Mellitus , Neoplasias Retroperitoneais , Feminino , Humanos , Adulto , Diabetes Insípido Neurogênico/diagnóstico , Diabetes Insípido Neurogênico/tratamento farmacológico , Diabetes Insípido Neurogênico/etiologia , Desamino Arginina Vasopressina/uso terapêutico , Neoplasias Retroperitoneais/complicaçõesRESUMO
BACKGROUND: Fucosylation is a crucial oligosaccharide modification in cancer and inflammation. Total cellular proteins of cancer cells and the sera of patients with cancer both show increased fucosylation levels. Certain kinds of fucosylated proteins can be applied as novel cancer biomarkers in glyco-proteomic analyses. We previously identified fucosylated haptoglobin (Fuc-Hpt) as a serologic marker for pancreatic cancer, and recently developed a lectin-antibody enzyme-linked immunosorbent assay system for quantifying Fuc-Hpt. In the present study, we investigated the clinical outcome of Fuc-Hpt levels in patients with colorectal cancer (CRC), and examined the mechanisms underlying Fut-Hpt production using a murine tumor transplantation model. METHODS: The relationship between Fuc-Hpt levels and clinical parameters was investigated in 77 patients with CRC, all of whom underwent primary resection. Serum Fuc-Hpt levels were examined in athymic nude mice injected with colon cancer cell lines that lacked fucosylation. RESULTS: Fuc-Hpt levels were significantly associated with overall and relapse-free survival, distant metastasis, clinical stage, and curability. Multivariate analysis revealed that distant metastasis was an independent factor for increased Fuc-Hpt levels. The combination of Fuc-Hpt and CEA might be a better serologic marker to predict prognosis. Fuc-Hpt levels were higher in mice with direct injection of tumor cells into the spleen than in those injected subcutaneously. CONCLUSIONS: Fuc-Hpt might be a useful marker for the prognosis of CRC. Fuc-Hpt could be produced by the tissue surrounding tumor cells, which might be the mechanism underlying Fuc-Hpt elevation associated with distant metastasis.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/sangue , Fucose/metabolismo , Haptoglobinas/análise , Idoso , Animais , Feminino , Haptoglobinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , PrognósticoRESUMO
The in vitro and in vivo inhibition of cytochrome P450 (CYP) 3A with mechanism-based inhibition (MBI) by macrolides was investigated using dexamethasone-treated female rats (DEX-female rats). In the in vitro CYP inhibition studies using erythromycin (ERM) and clarithromycin (CAM), similar inhibition responses were observed between human and DEX-female rat liver microsomes, however, there were fewer effects in intact male rats. The ex vivo study showed that midazolam (MDZ) metabolism in liver microsomes of DEX-female rats was reduced by ERM administration and the inhibitory effect was increased with increasing ERM doses, indicating that metabolite intermediate complex formation caused irreversible inhibition of CYP3A activity in DEX-female rats as well as in humans. In the in vivo studies, ERM and CAM significantly increased the area under the plasma concentration-time curve of MDZ and decreased the total clearance in DEX-female rats. It was concluded that the DDIs via MBI of CYP3A following macrolide administration in humans could be reproduced in female rats, suggesting that DEX-female rats can serve as an in vivo model for assessing this DDI in humans.
Assuntos
Claritromicina/farmacologia , Inibidores do Citocromo P-450 CYP3A , Eritromicina/farmacologia , Midazolam/metabolismo , Animais , Área Sob a Curva , Citocromo P-450 CYP3A/metabolismo , Dexametasona/administração & dosagem , Dexametasona/farmacologia , Interações Medicamentosas , Feminino , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Ratos , Fatores Sexuais , Especificidade da EspécieRESUMO
The aim of this study was to investigate the clinical course of masticatory function recovery following arthrocentesis. Patients with a unilateral condylar head fracture who underwent arthrocentesis for therapeutic reasons were evaluated and compared with patients with a unilateral condylar head fracture who did not undergo arthrocentesis. At 3 months after treatment, the occlusal contact area and maximum bite force in patients with a fracture treated with arthrocentesis were greater than in those who did not receive arthrocentesis at the same time points, although the differences were not significant. Moreover, at 1 and 3 months following arthrocentesis, mean (±SD) occlusal contact area (1 month: 1.99 ± 0.55 mm2, p = 0.01; 3 months: 2.90 ± 1.36 mm2, p = 0.03) and maximum bite force (1 month: 82.45 ± 15.04 N, p = 0.01; 3 months: 101.11 ± 14.53 N, p = 0.01) on the fractured side in patients who underwent that treatment were significantly reduced when compared with those on the non-fractured side. The authors conclude that if the priority is to avoid open reduction and internal fixation, then the arthrocentesis approach might be a less invasive alternative, albeit with the price of a prolonged healing interval.
Assuntos
Côndilo Mandibular , Fraturas Mandibulares , Artrocentese , Fixação Interna de Fraturas , Humanos , Côndilo Mandibular/cirurgia , Fraturas Mandibulares/cirurgia , Recuperação de Função FisiológicaRESUMO
Fluorescent proteins (FPs) have been widely used to visualize biological processes in living cells. It is essential to understand the underlying fluorescence mechanism to develop novel FPs and to interpret imaging data appropriately. Enhanced yellow fluorescent protein (eYFP) is one of the most typical FPs; however, several reports to date have been limited to individual discussion, which is insufficient to understand the full picture of the dynamics involved. In this study, we focused on the fluorescence resonance energy transfer (FRET) and dimerization behavior and performed picosecond time-resolved fluorescence measurements of eYFP and its A206K mutant, which does not form a dimer. The combination of the dissociation constant and the acid dissociation constant rationally explains the mechanism of ultrafast homo-FRET and ultrafast hetero-FRET. It is also shown that structural relaxation occurs in the dimer after excited-state proton transfer. The formation efficiencies and quaternary structures of dimers consisting of different protonation states are shown to be different. Furthermore, under high-concentration conditions, "slow" homo-FRET with tens of nanoseconds timescale occurs between monomers and dimers. The findings from this study will be applied to other fluorescent proteins such as Aequorea victoria green FP and its mutants and various red FPs with longer conjugation lengths.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Dimerização , Polarização de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismoRESUMO
Lignin is a phenylpropanoid polymer produced in the secondary cell walls of vascular plants. Although most eudicot and gymnosperm species generate lignins solely via polymerization of p-hydroxycinnamyl alcohols (monolignols), grasses additionally use a flavone, tricin, as a natural lignin monomer to generate tricin-incorporated lignin polymers in cell walls. We previously found that disruption of a rice 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (OsCAldOMT1) reduced extractable tricin-type metabolites in rice vegetative tissues. This same enzyme has also been implicated in the biosynthesis of sinapyl alcohol, a monolignol that constitutes syringyl lignin polymer units. Here, we further demonstrate through in-depth cell wall structural analyses that OsCAldOMT1-deficient rice plants produce altered lignins largely depleted in both syringyl and tricin units. We also show that recombinant OsCAldOMT1 displayed comparable substrate specificities towards both 5-hydroxyconiferaldehyde and selgin intermediates in the monolignol and tricin biosynthetic pathways, respectively. These data establish OsCAldOMT1 as a bifunctional O-methyltransferase predominantly involved in the two parallel metabolic pathways both dedicated to the biosynthesis of tricin-lignins in rice cell walls. Given that cell wall digestibility was greatly enhanced in the OsCAldOMT1-deficient rice plants, genetic manipulation of CAldOMTs conserved in grasses may serve as a potent strategy to improve biorefinery applications of grass biomass.
Assuntos
Parede Celular/metabolismo , Flavonoides/biossíntese , Lignina/biossíntese , Metiltransferases/metabolismo , Oryza/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
The CryoSeal® FS System has been recently introduced as an automated device for the production of complete fibrin glue from autologous plasma, rather than from pool allogenic or cattle blood, to prevent viral infection and allergic reaction. We evaluated the effectiveness of complete autologous fibrin glue and polyglycolic acid (PGA) sheet wound coverings in mucosa defect oral surgery. Postoperative pain, scar contracture, ingestion, tongue dyskinesia, and postoperative bleeding were evaluated in 12 patients who underwent oral (including the tongue) mucosa excision, and received a PGA sheet and an autologous fibrin glue covering. They were compared with 12 patients who received a PGA sheet and commercial allogenic fibrin glue. All cases in the complete autologous fibrin glue group demonstrated good wound healing without complications such as local infection or incomplete cure. All evaluated clinical measures in this group were similar or superior to the commercial allogenic fibrin glue group. Coagulation and adhesion quality achieved with this method was comparable to that with a PGA sheet and commercial fibrin glue. Covering oral surgery wounds with complete autologous fibrin glue produced by an automated device was convenient, safe, and reduced the risk of viral infection and allergic reaction associated with conventional techniques.
Assuntos
Bandagens , Adesivo Tecidual de Fibrina , Doenças da Boca/cirurgia , Boca/cirurgia , Ácido Poliglicólico , Adesivos Teciduais , Cicatrização , Autoenxertos , Curativos Biológicos , Humanos , Mucosa Bucal/cirurgia , Neoplasias Bucais/cirurgia , Procedimentos Cirúrgicos Bucais , Lesões Pré-Cancerosas/cirurgia , Infecção da Ferida Cirúrgica/prevenção & controle , Língua/cirurgiaRESUMO
UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K(m) value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6mM) of human liver microsomes, whereas V(max) values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K(m) (approximately 90 microM) or V(max) (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K(m) values (approximately 6mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V(max) value (0.1nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K(m) (210 microM) and V(max) (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K(m), 110 microM; V(max), 1.5nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.