RESUMO
INTRODUCTION: In the case reported here, the authors observed ovarian small cell carcinoma of the pulmonary type as it developed. CASE: The patient was a 48-year-old woman who underwent a hysterectomy for CIN3 in 2007. A year later, the woman underwent screening for ovarian cancer. A gradually growing ovarian mass was noted. This mass was found to be a mixed tumor. This mixed tumor grew to 36 mm in size, and six months later it had enlarged to 119 mm. After surgery, the tumor was pathologically diagnosed as an ovarian small cell carcinoma of the pulmonary type with a neuroendocrine nature that was positive for CD56 and synaptophysin. Postoperatively, the patient received six courses of combined therapy with irinotecan and cisplatin (CPT-P therapy), and the patient has survived disease- free for over two years. CONCLUSION: Findings suggested that ovarian small cell carcinoma of the pulmonary type is a type I ovarian malignancy that develops through an adenoma-carcinoma sequence.
Assuntos
Carcinoma de Células Pequenas/terapia , Neoplasias Ovarianas/terapia , Antígeno CD56/análise , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Carcinoma de Células Pequenas/patologia , Cisplatino/administração & dosagem , Terapia Combinada , Feminino , Humanos , Irinotecano , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologiaRESUMO
OBJECTIVES: This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation. METHODS: Injured human ACL tissues were harvested from 105 patients undergoing primary ACL reconstruction and divided into four phases based on the period from injury to surgery. Phase I was < three weeks, phase II was three to eight weeks, phase III was eight to 20 weeks, and phase IV was ≥ 21 weeks. Gene expressions of these tissues were analysed in each phase by quantitative real-time polymerase chain reaction using selected markers (collagen types 1 and 3, biglycan, decorin, α-smooth muscle actin, IL-6, TGF-ß1, MMP-1, MMP-2 and TIMP-1). Immunohistochemical staining was also performed using primary antibodies against CD68, CD55, Stat3 and phosphorylated-Stat3 (P-Stat3). RESULTS: Expression of IL-6 was mainly seen in phases I, II and III, collagen type 1 in phase II, MMP-1, 2 in phase III, and decorin, TGF-ß1 and α-smooth muscle actin in phase IV. Histologically, degradation and scar formation were seen in the ACL remnant after phase III. The numbers of CD55 and P-Stat3 positive cells were elevated from phase II to phase III. CONCLUSIONS: Elevated cell numbers including P-Stat3 positive cells were not related to collagens but to MMPs' expressions.
RESUMO
We investigated the effect of locally administered bisphosphonate on distraction osteogenesis in a rabbit model and evaluated its systemic effect. An osteotomy on the right tibia followed by distraction for four weeks was performed on 47 immature rabbits. They were divided into seven equal groups, with each group receiving a different treatment regime. Saline and three types of dosage of alendronate (low, 0.75 microg/kg; mid, 7.5 microg/kg and high 75 microg/kg) were given by systemic injection in four groups, and saline and two dosages (low and mild) were delivered by local injection to the distraction gap in the remaining three groups. The injections were performed five times weekly during the period of distraction. After nine weeks the animals were killed and image analysis and mechanical testing were performed on the distracted right tibiae and the left tibiae which served as a control group. The local low-dose alendronate group showed a mean increase in bone mineral density of 124.3 mg/cm(3) over the local saline group (analysis of variance, p < 0.05) without any adverse effect on the left control tibiae. The findings indicate that the administration of local low-dose alendronate could be an effective pharmacological means of improving bone formation in distraction osteogenesis.
Assuntos
Alendronato/administração & dosagem , Conservadores da Densidade Óssea/administração & dosagem , Osteogênese por Distração/métodos , Osteotomia/métodos , Animais , Modelos Biológicos , CoelhosRESUMO
OBJECTIVE: Inflammatory cytokines have been reported to be related with inflammation and expansion of jaw cysts. In this study, to examine the relationship between radicular cysts and inflammatory cytokines, it was found that there was notable unique evidence on cytokine synthesis from fibroblasts isolated from radicular cysts. METHODS: The expression of such cytokines, namely, interleukin-1beta, IL-1beta, IL-6, IL-8, IL-10, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta1 (TGF-beta1), and granulocyte-macrophage colony-stimulating (GM-CSF) mRNA, in nine radicular cysts was examined and compared with that detected in six specimens of healthy gingival mucosa. Furthermore, separating all fibroblasts from their respective radicular cysts, healthy gingival mucosa, and healthy periodontal ligaments, these fibroblast groups were cultured without stimulators and a supernatant for each was obtained to analyse IL-1beta, IL-6, IL-8, TNF-alpha, and IFN-gamma by ELISA. RESULTS: Differences between radicular cysts and healthy gingival mucosa were not clearly shown by the expression of cytokine mRNA. Analysing inflammatory cytokine synthesis in fibroblast groups from these three kinds of tissues, surprisingly, the levels of IL-6 mRNA and protein were recognised to be higher in fibroblasts of radicular cysts than in those of control tissues by ELISA and a real-time RT-PCR. Significant differences in the cultured supernatants of these fibroblast groups were not recognised in the release of IL-1beta, IL-8, TNF-alpha, and IFN-gamma by ELISA. CONCLUSIONS: From these results, it was suggested that fibroblasts inducing IL-6 production might play important roles in the expansion of radicular cysts. It is considered that fibroblasts around radicular cysts may lead to high IL-6 synthesis over time in chronic inflammation.
Assuntos
Fibroblastos/metabolismo , Doenças da Gengiva/metabolismo , Interleucina-6/biossíntese , Cisto Radicular/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Doença Crônica , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Gengiva/metabolismo , Substâncias de Crescimento/genética , Substâncias de Crescimento/metabolismo , Humanos , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin beta1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin beta1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.
Assuntos
Ligamentos Longitudinais/metabolismo , Proteínas de Neoplasias/biossíntese , Ossificação do Ligamento Longitudinal Posterior/metabolismo , Ossificação Heterotópica/metabolismo , Estresse Mecânico , Fatores de Transcrição/biossíntese , Idoso , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/efeitos dos fármacos , Butadienos , Células Cultivadas , Colágeno Tipo I/biossíntese , Colágeno Tipo I/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Cadeias beta de Integrinas/biossíntese , Cadeias beta de Integrinas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Nitrilas , Ossificação do Ligamento Longitudinal Posterior/fisiopatologia , Osteocalcina/biossíntese , Osteocalcina/efeitos dos fármacos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Recent mucosal biopsy criteria combined with endoscopy effectively differentiate patients with ulcerative colitis ultimately requiring surgery (UC-S) from those receiving medication alone (UC-M). However, the criteria were inconvenient in practical use because of the need for complicated calculations, and the validity has not been verified in other institutes where the indications for surgery may differ. The aims of this multicenter study were to propose simple criteria in which calculation can be performed by mental arithmetic and to measure their validity. METHODS: Based on the above original criteria, we constructed simple criteria in which coefficients and constant were simplified to integral numbers. The criteria consisted of the diagnostic categories, highest-risk, high-risk, unpredictable, low-risk, and lowest-risk of surgery. The validity of these proposed criteria was evaluated in 121 patients with UC-S and 186 with UC-M from 11 institutes. RESULTS: The categories of high-risk and low-risk had sensitivities exceeding 86.0% and specificities exceeding 95.2%, and the validities were maintained at high levels in most individual institutes. There was little difference in validity between the proposed and original criteria when testing using the same patients. CONCLUSIONS: Despite simplified coefficients and constant, the proposed criteria reliably predicted the eventual clinical outcome of patients with ulcerative colitis and would be helpful in determining the necessity of surgery.
Assuntos
Colectomia/métodos , Colite Ulcerativa/patologia , Colite Ulcerativa/cirurgia , Colonoscopia/métodos , Mucosa Intestinal/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The clinical course of patients with ulcerative colitis (UC) is unpredictable, and 17%-38% ultimately require surgery. We hypothesized that mucosal histology may differ between patients requiring surgery and those receiving medication alone. The aim of this study was to elucidate comprehensive criteria consisting of specific histologic features enabling the prediction of failure to medical treatment. METHODS: We studied colorectal biopsy specimens from 67 patients ultimately requiring surgery (UC-S) and 90 receiving medication alone for more than 3 years (UC-M), and conducted multiple logistic regression analysis on 70 histologic features together with endoscopic disease extent and patient age. The analysis constructed an equation finding probability of UC-S (P(UC-S)). Based on a receiver-operating characteristic curve, we selected four cut-off values of P(UC-S), and determined criteria of five categories: highest-risk, higher-risk, unpredictable, lower-risk and lowest-risk of surgery. Sensitivity and specificity of criteria were evaluated in a 2 x 5 table. RESULTS: Statistically significant features predicting UC-S were deep ulceration (X1), frequent crypt abscesses (X2), focal and segmental mononuclear cell infiltration (X3 and X4), paucity of eosinophils (X5: eosinophil infiltration) and wide extent of the disease (X6). The regression equation was as follows: logitP(UC-S) = -16.26 + 3.20X1 + 4.83X2 + 11.65X3 + 5. 10X4 - 5.59X5 + 5.53X6. Higher-risk and lower-risk showed sensitivity exceeding 91.0% and specificity exceeding 98.5% in predicting the outcome. CONCLUSIONS: Our criteria incorporating specific histologic features and endoscopic disease extent reliably predict eventual clinical outcome, and are expected to prove useful in determining the necessity of surgery.
Assuntos
Colite Ulcerativa/parasitologia , Colo/patologia , Adulto , Biópsia , Estudos de Casos e Controles , Colite Ulcerativa/cirurgia , Feminino , Humanos , Modelos Logísticos , Masculino , Valor Preditivo dos Testes , Curva ROC , Reto/patologia , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
It is a recognized fact that glandular structures sometimes occur in peripheral nerve sheath tumors (PNST). Reports indicate that epithelial potential could be expressed in malignant PNST, while the glands in most benign PNST could be trapped skin adnexa. We present a case of spindle cell tumor with glandular structures. The patient was a 55-year-old man who had a subcutaneous tumor excised. The spindle cell tumor had histological characteristics of neurofibroma. The glandular structures had a pattern of immunohistochemical staining that was similar to that of the secretory coils and excretory ducts of normal eccrine glands. Therefore, the glands were thought to be naturally existing eccrine glands. The glands were completely enclosed within the tumor and not connected to one another; a cluster formation was not observed. The upper portion of tumor included some glands of skin adnexa. The spindle cell tumor may have originated from the nerves distributed around the skin adnexa, and grown to the subcutaneous tissue. The glands may have been left behind rather than have been trapped in the tumor.
Assuntos
Neoplasias de Bainha Neural/etiologia , Neoplasias de Bainha Neural/patologia , Glândulas Écrinas/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
We described histologic changes in patellar tendon autografts that occur over time within the tibial tunnel in specimens harvested from patients undergoing revision anterior cruciate ligament reconstruction. Ten patients, averaging 21.2 years of age, were divided into two groups based on the time period between their original and revision surgery: early revision (less than 1 year, four patients) and late revision (more than 1 year, six patients). Among the early revision group, the tendon within the tunnel showed increased cellularity and random collagen bundles. A specimen from the shortest early revision case revealed a normal original bone-tendon junction, whereas others showed an obscured structure. Between the tendon and the tunnel wall, granulation tissue was seen and the bone-tendon junction was still immature. In the late revision group, the tendon appeared similar to normal ligament. The original bone-tendon junction was not seen, and the tendon continued completely to the tunnel wall with Sharpey-like fibers. Observations in the early revision group suggest that tendon remodeling and bone-tendon integration continue for at least several months after transplantation. The original bone-tendon junction appears to have shifted to the proximal patellar tendon-tunnel wall junction with time. These findings are in agreement with prior animal studies.
Assuntos
Ligamento Cruzado Anterior/cirurgia , Sobrevivência de Enxerto , Procedimentos de Cirurgia Plástica/métodos , Tendões/patologia , Tendões/transplante , Tíbia/cirurgia , Adolescente , Adulto , Feminino , Humanos , Instabilidade Articular/cirurgia , Traumatismos do Joelho/cirurgia , Masculino , Reoperação , Tíbia/patologia , Coleta de Tecidos e Órgãos/métodos , Transplante Autólogo , CicatrizaçãoRESUMO
Aureusidin synthase, which plays a key role in the yellow coloration of snapdragon flowers, is a homolog of plant polyphenol oxidase (PPO). The enzyme specifically acted on chalcones with a 4-monohydroxy or 3,4-dihydroxy B-ring to produce aurones, for whose production the oxidative cyclization of chalcones must be preceded by 3-oxygenation. However, it exhibited virtually no PPO activity toward non-chalcone phenolics. The enzyme was competitively inhibited by phenylthiourea, a specific PPO inhibitor. These results led us to propose a mechanism of aurone synthesis by aureusidin synthase on the basis of known PPO-catalyzed reactions and conclude that the enzyme is a chalcone-specific PPO specialized for aurone biosynthesis.
Assuntos
Benzofuranos/metabolismo , Catecol Oxidase/metabolismo , Magnoliopsida/enzimologia , Oxigenases de Função Mista/metabolismo , Pigmentação , Estruturas Vegetais/metabolismo , Benzofuranos/química , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/química , Chalcona/análogos & derivados , Chalcona/química , Chalcona/metabolismo , Chalconas , Inibidores Enzimáticos/farmacologia , Magnoliopsida/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Modelos Moleculares , Conformação Molecular , Estruturas Vegetais/enzimologia , Especificidade por SubstratoRESUMO
An osteosarcoma cell line has been established from a soft tissue tumor that occurred spontaneously in a BALB / c mouse. This cell line showed ossification when transplanted into syngeneic mice. To examine the mechanism of bone formation, the expression of mRNAs for osteoblastic and chondroblastic markers and factors associated with ossification has been investigated. In culture, the cells exhibited a spindle shape in the growth phase, but had a polygonal shape in the stationary phase. Reverse transcription-polymerase chain reaction analysis showed that the cells expressed mRNAs for pro-alpha1(I) chain of type I collagen, alkaline phosphatase, osteopontin, osteocalcin, and core binding factor alpha1, suggesting differentiation into the stage of osteoblasts during the stationary phase. After transplantation, histological examination revealed small foci of pale blue material and basophilic networks that were scattered in the tumor tissues at one week. The former stained positive with alcian blue, suggesting a chondroid matrix. Pro-alpha1(II) chain of type II collagen mRNA was expressed at one week. A large part of tumors at two and three weeks consisted of basophilic networks, which stained positive via von Kossa's method, indicating a calcified woven bone. In situ hybridization analysis showed strong expression of osteopontin and osteocalcin mRNAs in tumor cells surrounding the bone matrix. Bone morphogenetic protein-6 and -7 mRNAs were detected in transplanted tumors, but not in cultured cells. These results suggest that the cell line has the properties of an osteoblastic lineage when cultured in vitro and has an ossifying ability through endochondral bone formation processes when transplanted in vivo.
Assuntos
Osteossarcoma/patologia , Transplante , Células Tumorais Cultivadas , Fosfatase Alcalina/biossíntese , Animais , Desenvolvimento Ósseo/fisiologia , Técnicas de Cultura de Células/métodos , Subunidades alfa de Fatores de Ligação ao Core , Proteínas de Ligação a DNA/biossíntese , Extremidades/diagnóstico por imagem , Feminino , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Osteocalcina/biossíntese , Osteopontina , Pró-Colágeno/biossíntese , RNA Mensageiro/metabolismo , Radiografia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/biossíntese , Neoplasias de Tecidos Moles/patologia , Fatores de Tempo , Fatores de Transcrição/biossínteseRESUMO
We present a case of intra-epithelial carcinoma occurring in a serrated adenoma of the colon. The pedunculated polyp, which measured 12 x 10 x 6 mm, was endoscopically removed from the ascending colon of a 78-year-old woman. Histologically, the polyp mainly consisted of serrated adenomatous glands, and had foci of intra-epithelial carcinoma at the top. Hyperplastic (metaplastic) areas were also present in both borders between the serrated adenomatous area and the surrounding normal mucosa. A sequential increase in the degree of dysplasia, and in the number of nuclei positively reactive for Ki-67 and p53 was evident from the hyperplastic areas toward the foci of carcinoma. The polyp described here may represent a carcinogenic potential of hyperplastic polyp via serrated adenoma.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Adenocarcinoma/metabolismo , Adenoma/metabolismo , Idoso , Pólipos do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Neoplasias Primárias Múltiplas , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND AND AIM: Colorectal Paneth cell metaplasia (PCM) is known to be a sign of idiopathic inflammatory bowel disease (IBD), although its distribution and histogenesis are not fully understood. Objectives of this research were to investigate the spatial distribution of PCM in IBD and other forms of colitis (non-IBD), and to find stimuli causing PCM. METHODS: We studied multiple biopsy specimens from 181 patients with ulcerative colitis (UC), 159 with Crohn's disease (CD), 448 with non-IBD, and 78 normal controls. Paneth cell metaplasia frequency, at each colorectal site, was evaluated to find possible differences among diseases, phases of activity, and extents of disease. RESULTS: In non-IBD and controls, PCM was rarely (0-1.9%) seen at distal sites, but frequently (up to 48.7%) found at the ascending colon and cecum (P < 0.001). Paneth cell metaplasia frequency was significantly higher in IBD than in non-IBD patients and controls at distal sites (P < 0.001), but did not differ significantly between UC and CD, or among active, resolving, and quiescent phases. In UC, proctitis and left-sided colitis rarely displayed PCM at unaffected sites. Multiple logistic regression analysis revealed that PCM was positively associated with crypt distortion and mononuclear cell infiltration (P < 0.005), but negatively or not significantly associated with crypt atrophy, mucin depletion, acute inflammation, or phase of activity. CONCLUSIONS: Paneth cell metaplasia is a non-specific phenomenon in the proximal colon, but distal PCM, which occurs exclusively in affected mucosa, is a useful marker indicating IBD, even in the inactive phase. Regression analysis suggests that repair and regeneration may be the most potent stimuli causing PCM.
Assuntos
Colite Ulcerativa/patologia , Doença de Crohn/patologia , Celulas de Paneth/patologia , Adulto , Biópsia/métodos , Colite Ulcerativa/diagnóstico , Doença de Crohn/diagnóstico , Humanos , Metaplasia/patologia , Pessoa de Meia-Idade , Análise de RegressãoRESUMO
BACKGROUND AND AIM: Simple mucosal biopsy criteria proposed by authors reliably differentiate idiopathic inflammatory bowel disease (IBD) from other forms of colitis (non-IBD) and Crohn's disease involving the colon (CD) from ulcerative colitis (UC). The aim of this study is to investigate the reproducibility of these criteria. METHODS: Three established pathologists and two medical students blindly examined 20 sets of multiple biopsy slides from patients with CD, 20 from those with UC, and 20 from those with non-IBD. The students had been given instructions on histological definitions using another 15 sets previously. Each observer evaluated 10 histological items required in the criteria and determined categorical diagnoses such as definite IBD and probable UC. Interobserver agreement for the individual histological items was measured by using kappa analysis and Pearson's correlation, while it was measured for categorical diagnoses with the use of Spearman's rank correlation. RESULTS: All of the individual histological items expressed excellent or fair-to-good agreement among the five observers, although two items associated with the criteria for CD and UC had poor agreements among the students. With regard to categorical diagnoses based on the criteria for IBD and non-IBD, and those for CD and UC, coefficients for Spearman's rank correlation exceeded 0.92 and 0.86 among the pathologists, and 0.76 and 0.74 among the students, respectively; all of the coefficients were statistically significant (P < 0.05). CONCLUSIONS: The simple criteria were sufficiently reproducible and would help most pathologists to make an automated and objective diagnosis.
Assuntos
Colite Ulcerativa/patologia , Doença de Crohn/patologia , Biópsia/métodos , Colite/patologia , Diagnóstico Diferencial , Humanos , Mucosa Intestinal/patologia , Variações Dependentes do Observador , Sensibilidade e Especificidade , Estatísticas não ParamétricasRESUMO
A new tetracyclic terpenoid hydrocarbon, cyclosinularane (1), and (+)-alloaromadendrene (2), the enantiomer of terrestrial (-)-alloaromadendrene, have been isolated from the volatile oil of the soft corals Clavularia viridis and Sarcophyton acutangulum, respectively. Their structures have been determined on the basis of NMR spectral analysis and their chiroptical properties.
Assuntos
Cnidários/química , Óleos/química , Terpenos/química , Animais , Artemia , Azulenos , Ensaios de Seleção de Medicamentos Antitumorais , Dose Letal Mediana , Espectroscopia de Ressonância Magnética , Conformação Molecular , Sesquiterpenos , Terpenos/isolamento & purificação , Terpenos/toxicidade , Células Tumorais CultivadasRESUMO
Aurones are plant flavonoids that provide yellow color to the flowers of some popular ornamental plants, such as snapdragon and cosmos. In this study, we have identified an enzyme responsible for the synthesis of aurone from chalcones in the yellow snapdragon flower. The enzyme (aureusidin synthase) is a 39-kilodalton, copper-containing glycoprotein catalyzing the hydroxylation and/or oxidative cyclization of the precursor chalcones, 2',4',6',4-tetrahydroxychalcone and 2',4',6',3,4-pentahydroxychalcone. The complementary DNA encoding aureusidin synthase is expressed in the petals of aurone-containing varieties. DNA sequence analysis revealed that aureusidin synthase belongs to the plant polyphenol oxidase family, providing an unequivocal example of the function of the polyphenol oxidase homolog in plants, i.e., flower coloration.
Assuntos
Benzofuranos/metabolismo , Magnoliopsida/enzimologia , Sequência de Aminoácidos , Catálise , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Ciclização , DNA Complementar , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Precursores Enzimáticos/metabolismo , Genes de Plantas , Hidroxilação , Magnoliopsida/genética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Peso Molecular , Pigmentação , Estruturas Vegetais/enzimologia , Plantas/enzimologia , Homologia de Sequência de AminoácidosRESUMO
We have isolated and characterized a cDNA, PfAT208, encoding hydroxycinnamoyl-CoA: anthocyanin 3-O-glucoside-6"-O-acyltransferase (3AT) from Perilla frutescens. The identity of the cDNA was established by determination of the reaction products with recombinant enzyme overexpressed in Escherichia coli. The deduced amino acid sequence has a few regions that are conserved in a CoA-dependent acyltransferase family. The recombinant enzyme produced in yeast could utilize cyanidin 3-glucoside and cyanidin 3,5-diglucoside, putative substrates in vivo, as well as other anthocyanins. The inhibitory effects of diethyl pyrocarbonate and N-ethylmaleimide on the recombinant 3AT activities suggest that histidine and cysteine residues are important for their catalytic function. These properties are in common with anthocyanin 5-O-glucoside-6"-O-acyltransferase (5AT). In Northern analysis, a transcript of PfAT208 was detected in the young leaves of perilla red forma. The properties of other cDNAs, gentian GAT106 and petunia PhAT48, isolated during the above cloning procedure are also described.
Assuntos
Aciltransferases/genética , Antocianinas/biossíntese , Ácidos Cumáricos , Lamiaceae/genética , Acil Coenzima A/metabolismo , Aciltransferases/efeitos dos fármacos , Aciltransferases/metabolismo , Sequência de Aminoácidos , Antocianinas/metabolismo , Clonagem Molecular , Sequência Conservada , Dietil Pirocarbonato/farmacologia , Etilmaleimida/farmacologia , Glucosídeos/metabolismo , Lamiaceae/enzimologia , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor. Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems. Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots. The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively. The molar ratio of Fd III to SiR in root plastids was approximately 3:1. Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR. In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I. Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested. NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner. These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction. In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I.
Assuntos
Ferredoxinas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Zea mays/metabolismo , Transporte de Elétrons , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Teste de Complementação Genética , Mutação , NADP/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Fotossíntese , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Zea mays/genéticaRESUMO
It has been shown that urea in fermented beverages and foods can serve as a precursor of ethylcarbamate, a potential carcinogen, and acid urease is an effective agent for removing urea in such products. We describe herein the purification and characterization of a novel acid urease from Arthrobacter mobilis SAM 0752 and show its unique application for the removal of urea from fermented beverages using the Japanese rice wine, sake, as an example. The purified acid urease showed an optimum pH for activity at pH 4.2. The enzyme exhibited an apparent K(m) for urea of 3.0 mM and a Vmax of 2370 mumol of urea per mg and min at 37 degrees C and pH 4.2. Gel permeation chromatographic and sodium dodecyl sulfate gel electrophoretic analyses showed that the enzyme has an apparent native molecular weight (M(r)) of 290,000 and consisted of three types of subunit proteins (M(r), 67,000, 16,600, 14,100) denoted by alpha, beta, and gamma. The most probable stoichiometry of the subunits was estimated to be alpha: beta: gamma = 1:1:1, suggesting the enzyme subunit structure of (alpha beta gamma)3. The enzyme also existed as an aggregated form with an M(r) of 580,000. The purified enzyme contained 2 g-atom of nickel per alpha beta gamma unit of the enzyme. Enzyme activity was inhibited by acetohydroxamic acid, HgCl2, and CuCl2. The isoelectric point of the native enzyme was estimated by gel electrofocusing to be 6.8. Urea (50 ppm), which was exogenously added to sake (pH 4.4, 17 +/- 1% (v/v) ethanol), was completely decomposed by incubation with the enzyme (0.09 U ml-1) at 15 degrees C for 13 days. The enzyme was unstable at temperatures higher than 65 degrees C and pHs lower than 4, and was completely inactivated under the conditions of a pasteurization step involved in the traditional sake-making processes. These results indicate that the enzyme is applicable to the elimination of urea in fermented beverages with minimal modification to the conventional process.