Assuntos
Aminoácido Oxirredutases/farmacologia , Antifibróticos/farmacologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/normas , Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/uso terapêutico , Antifibróticos/análise , Antifibróticos/uso terapêutico , Técnicas Biossensoriais/métodos , Fibrose/tratamento farmacológico , Fibrose/prevenção & controle , HumanosRESUMO
In recent years, breakthroughs in the field of tumor immunotherapy with immune checkpoint inhibitors (ICIs) have made a therapeutic revolution, which has been shown to improve the prognosis of patients with hepatocellular carcinoma (HCC). Immune infiltrates represent a major component of tumor microenvironment (TME), and play an essential role in both tumor progression and therapeutic response. The major unmet challenge in tumor immunotherapy is exploring the intrinsic and extrinsic mechanisms of TME promoting the management of HCC. Lysyl oxidase like 3 (LOXL3) participates in the remodeling of extracellular matrix (ECM) and the cross-linking of collagen and elastic fibers. It has been reported that LOXL3 is associated with the development and tumorigenesis of multiple types of cancer. RNA sequencing data and corresponding clinical information were extracted from The Cancer Genome Atlas (TCGA) databases, then subjected to gene expression, tumor microenvironment, survival, enrichment analyses utilizing R packages. In this study, we first found that LOXL3 gene was upregulated in tumor tissues compared with the normal tissues. Furthermore, LOXL3 expression is positively correlated with the infiltration of multiple immune cells and the expression of immune checkpoint genes in HCC. Meanwhile, high LOXL3 expression predicted poor outcomes of the patients with HCC. Functional enrichment analysis suggested that LOXL3 was mainly linked to extracellular structure and matrix organization, cell-cell adhesion, and T cell activation. This is the first comprehensive study to indicate that LOXL3 is correlated with immune infiltrates and may serve as a novel biomarker predicting prognosis and immunotherapy in HCC.
Assuntos
Aminoácido Oxirredutases/análise , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Linfócitos do Interstício Tumoral/enzimologia , Microambiente Tumoral , Aminoácido Oxirredutases/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Dano ao DNA , Metilação de DNA , Reparo do DNA , Bases de Dados Genéticas , Humanos , Proteínas de Checkpoint Imunológico/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Linfócitos do Interstício Tumoral/imunologia , Prognóstico , Regulação para CimaRESUMO
The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme-substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.
Assuntos
Aminoácido Oxirredutases/análise , Técnicas Biossensoriais , Hypocreales , Eletrodos , Enzimas Imobilizadas , Concentração de Íons de Hidrogênio , Cinética , LisinaRESUMO
In this paper, we present a simple and label-free colorimetric biosensor for detection of the nopaline synthase (NOS) terminator in genetically modified (GM) plants. The "signal on" colorimetric biosensor was developed using a nanocomposite consisted of gold nanoparticles doped magnetic Fe3O4 nanoparticles (Fe3O4@Au NP), capture probe DNA (cDNA), and hemin-functionalized reduced graphene oxide nanosheets (H-GN). The nanocomposite was successfully prepared by means of Au-S bonds and the strong π interactions between cDNA and H-GN. The sensing approach is based on the excellent peroxidase-mimicking activity of H-GN and its different electrostatic interactions with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In presence of the target NOS, the cDNA in the nanocomposite will hybridize with its complementary sequence, and form dsDNA structure. Due to the weak π interactions between dsDNA and H-GN, a portion of H-GN will be released from the surface of Fe3O4@Au NPs and transferred into solution. After magnetic separation was performed, the supernatant was incubated with 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The released H-GN can catalyze the oxidation reaction of TMB and turn the colorless solution blue. This "signal-on" colorimetric biosensor shows a broad linear range of 0.5-100 nM for the target NOS, with a 0.19 nM detection limit. The application of the biosensor for determination of NOS segments in samples of GM and non-GM tomatoes shows that it can discriminate between GM and non-GM plants. The reliability of the method for samples of NOS-spiked GM tomato suggests satisfactory recoveries in the range of 93.6%-94.2%.
Assuntos
Aminoácido Oxirredutases/análise , Técnicas Biossensoriais , Colorimetria , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Compostos Férricos/química , Ouro/química , Grafite/química , Hemina/química , Humanos , Oxirredução , Tamanho da Partícula , Reação em Cadeia da Polimerase , Propriedades de SuperfícieAssuntos
Hipertensão Induzida pela Gravidez/fisiopatologia , Pré-Eclâmpsia/fisiopatologia , Complicações Cardiovasculares na Gravidez/fisiopatologia , Adulto , Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/sangue , Contagem de Células Sanguíneas/métodos , Creatinina/análise , Creatinina/sangue , Feminino , Humanos , Hipertensão Induzida pela Gravidez/classificação , Gravidez , Proteinúria/urina , Ácido Úrico/análise , Ácido Úrico/sangueRESUMO
Extracellular matrix (ECM) macromolecules, apart from structural role for the surrounding tissue, have also been defined as crucial mediators in several cell mechanisms. The proteolytic and cross-linking cascades of ECM have fundamental importance in health and disease, which is increasingly becoming acknowledged. However, formidable challenges remain to identify the diverse and novel role of ECM molecules, especially with regard to their distinct biophysical, biochemical, and structural properties. Considering the heterogeneous, dynamic, and hierarchical nature of ECM, the characterization of 3D functional molecular view of ECM in atomic detail will be very useful for further ECM-related studies. Nowadays, the creation of a pioneer ECM multidisciplinary integrated platform in order to decipher ECM homeostasis is more possible than ever. The access to cutting-edge technologies, such as optical imaging and electron and atomic force microscopies, along with diffraction and X-ray-based spectroscopic methods can integrate spanning wide ranges of spatial and time resolutions. Subsequently, ECM image-guided site-directed proteomics can reveal molecular compositions in defined native and reconstituted ECM microenvironments. In addition, the use of highly selective ECM enzyme inhibitors enables the comparative molecular analyses within pre-classified remodeled ECM microenvironments. Mechanistic information which will be derived can be used to develop novel protein-based inhibitors for effective diagnostic and/or therapeutic modalities targeting ECM reactions within tissue microenvironment.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteômica/métodos , Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/metabolismo , Animais , Descoberta de Drogas/métodos , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/análise , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proteína-Lisina 6-Oxidase/análise , Proteína-Lisina 6-Oxidase/metabolismo , ProteóliseRESUMO
BACKGROUND: People with HIV are at for metabolic syndrome (MetS) and fatty liver disease, but the role of Antiretroviral therapy (ART) is poorly understood. MetS and fatty liver disease been associated with changes in adiponectin, soluble ST2 (sST2), chitinase 3-like 1 (Chi3L1), hyaluronic acid (HA), tissue inhibitor of metalloproteinase-1 (TIMP-1), lysyl oxidase-like-2 (LOXL2) and transforming growth factor ß (TGF-ß) concentrations in HIV-uninfected populations. Protease (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) may contribute to these comorbidities, but the effects of switching from PI- or NNRTI to raltegravir (RAL) on these biomarkers is unknown. METHODS: Cryopreserved plasma was obtained from a completed, prospective trial of HIV-infected women with central adiposity on NNRTI- or PI-based ART during which they were randomized to remain on their current ART or switch to a RAL based regimen. Biomarker concentrations were quantified using ELISA and Multiplex assays at baseline and 24 weeks after randomization. Wilcoxon-signed rank test evaluated within-group changes, Spearman and linear regression models evaluated correlations between biomarkers and clinical covariates. RESULTS: Participants had a median age of 43 years, CD4+ T lymphocyte count 558 cells/mm3 and BMI 32 kg/m2; 35% met criteria for MetS. At baseline, higher adiponectin levels correlated with higher Chi3L1 levels (r = 0.42, p = 0.02), as did declines after 24 weeks (r = 0.40, p = 0.03). Changes in sST2 correlated with changes in Chi3L1 (r = 0.43, p = 0.02) and adiponectin (r = 0.40, p = 0.03). Adiponectin and Chi3L1 levels decreased significantly in women switched to RAL vs continue PI/NNRTI. CONCLUSION: In women with HIV and central obesity, the hepatic steatosis/fibrosis marker Chi3L1 and adiponectin decrease in conjunction with sST2 decreases following switch to RAL. Whether switching from NNRTI/PI-based regimens to RAL can improve hepatic steatosis and dysmetabolism requires further study. TRIAL REGISTRATION: Clinicaltrials.gov NCT00656175.
Assuntos
Adiponectina/uso terapêutico , Proteína 1 Semelhante à Quitinase-3/efeitos dos fármacos , Raltegravir Potássico/farmacologia , Adiponectina/sangue , Adiponectina/metabolismo , Adiponectina/farmacologia , Adulto , Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/sangue , Fármacos Anti-HIV/uso terapêutico , Biomarcadores/sangue , Contagem de Linfócito CD4 , Proteína 1 Semelhante à Quitinase-3/metabolismo , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Feminino , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/uso terapêutico , Humanos , Síndrome Metabólica/metabolismo , Pessoa de Meia-Idade , Obesidade/fisiopatologia , Estudos Prospectivos , Raltegravir Potássico/metabolismo , Raltegravir Potássico/uso terapêutico , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/uso terapêutico , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/sangue , Carga ViralRESUMO
Tubulointerstitial fibrosis is a common end point of chronic kidney diseases, and preventing its progression is key to avoiding renal failure. Transforming growth factorß (TGFß) and associated molecules promote tubulointerstitial fibrosis; however, effective therapies targeting these molecules have yet to be developed. Lysyl oxidaselike 2 (LOXL2), which is involved in invasive growth and metastasis of malignant neoplasms, has recently been reported to serve a key role in hepatic and pulmonary fibrosis. However, little is currently known regarding LOXL2 expression in the kidney and its involvement in tubulointerstitial fibrosis. The present study evaluated LOXL2 expression in human and mouse kidney tissues, as well as in cultured renal cells. LOXL2 protein expression was detected in glomerular capillary loops and tubular epithelial cells in human and mouse kidneys. Glomerular LOXL2 was localized to the cytoplasm of podocytes, as determined by double immunofluorescence microscopy using a podocyte marker (synaptopodin). This result was supported by western blot analysis, which demonstrated that LOXL2 protein expression is present in cultured human podocytes and HK2 human proximal tubular cells. In addition, the mRNA and protein expression levels of LOXL2 were higher in a mouse model of tubulointerstitial fibrosis compared with in control mice. In addition, immunohistochemistry results demonstrated that LOXL2 is present in the fibrous interstitium and infiltrating mononuclear cells in a mouse model of tubulointerstitial fibrosis. The present study demonstrated that LOXL2 is expressed in compartments of renal tissue, where it appears to contribute to the progression of tubulointerstitial fibrosis.
Assuntos
Aminoácido Oxirredutases/análise , Túbulos Renais/patologia , Rim/patologia , Insuficiência Renal Crônica/patologia , Aminoácido Oxirredutases/genética , Animais , Linhagem Celular , Progressão da Doença , Fibrose , Regulação da Expressão Gênica , Humanos , Rim/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/metabolismo , Masculino , Camundongos , Podócitos/metabolismo , Podócitos/patologia , Insuficiência Renal Crônica/genéticaRESUMO
The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5'- nucleotidases (5'-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms. The major differences in the venom compositions are the higher concentration of SVMPs and CRISPs in the P. butleri venom, as well as the presence of post-synaptic neurotoxins. Furthermore, the analysis revealed a high concentration of proteins with myotoxic, coagulopathic and apoptotic activities. PLA2s are responsible for the myotoxic and anticoagulant effects observed in patients after envenomation (4). The other protein families, encountered in the two venoms, probably contribute to the major symptoms described for these venoms. These results explain the observed clinical effects of the black snake envenomation. The analyzed venoms contain group P-III metalloproteinases of medical importance with the potency to be used for diagnostic purposes of von Willebrand factor (vWF) disease, for regulation of vWF in thrombosis and haemostasis, for studying the function of the complement system in host defense and in the pathogenesis of diseases. Comparison of venomic data showed similarities in the major venom components of snakes from the genus Pseudechis, resulting in common clinical effects of envenomation, and demonstrating close relationships between venom toxins of Elapidae snakes.
Assuntos
Peptídeos/análise , Proteoma/análise , Venenos de Serpentes/metabolismo , Serpentes/metabolismo , Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/metabolismo , Animais , Austrália , Eletroforese em Gel de Poliacrilamida , Metaloendopeptidases/análise , Metaloendopeptidases/metabolismo , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/metabolismo , Peptídeos/metabolismo , Fosfolipases A2/análise , Fosfolipases A2/metabolismo , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: This study aimed to compare cellular expression of lysyl oxidase-like 1 (LOXL1), a key enzyme in elastin metabolism, of premenopausal women with pelvic organ prolapse (POP) compared with premenopausal controls without POP and postmenopausal women with POP. In addition, we examined whether variation of LOXL1 expression was dependent on biopsy site. METHODS: A standardized protocol was utilized to obtain vaginal biopsies from 30 women (10 premenopausal POP, 10 postmenopausal POP, and 10 premenopausal non-POP). Expression levels of messenger RNA (mRNA) and protein of LOXL1 were determined using real-time quantitative polymerase chain reactions and enzyme-linked immunosorbant assays. Analysis was performed to determine if there were differences between group or biopsy site. RESULTS: Significant differences in LOXL1 mRNA expression were found between patient groups (P = 0.0033). LOXL1 mRNA expression (relative to 18S) was upregulated in the postmenopausal POP group (54.5 ± 14.7) compared with the premenopausal POP group (5.2 ± 14.7, P = 0.0034) and the premenopausal non-POP group (23 ± 18, P = 0.0359). No significant differences in LOXL1 protein expression (nanogram/milliliter per microgram total protein) were seen between groups (premenopausal POP, 3.2 × 10 ± 6.3 × 10; postmenopausal POP, 4.3 × 10 ± 6.3 × 10; premenopausal non-POP, 5.0 × 10 ± 7.7 × 10; P = 0.15). No differences in mRNA expression were seen between sites (P = 0.74), but significant variation was noted in protein expression (P = 0.001). CONCLUSIONS: Premenopausal and postmenopausal women with POP exhibit differential expression of LOXL1 suggesting different pathways in the pathogenesis of POP. The role of biopsy location on LOXL1 expression requires further investigation.
Assuntos
Aminoácido Oxirredutases/metabolismo , Prolapso de Órgão Pélvico/enzimologia , Pós-Menopausa/metabolismo , Pré-Menopausa/metabolismo , Adulto , Aminoácido Oxirredutases/análise , Biópsia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Vagina/patologiaRESUMO
BACKGROUND: 67 laminin receptor (67LR) plays an important role in the invasion and metastasis of cholangiocarcinoma, but its mechanism remains unclear. AIMS: We investigated the clinical significance of 67LR and its relation to lysyl oxidase-like 2 (LOXL2) in 67LR-mediated invasion and metastasis in cholangiocarcinoma. METHODS: The clinical significance of 67LR and LOXL2 expression and the prognosis of patients were investigated in 73 cancerous and 32 paracancerous tissues by immunohistochemistry. The impact of LOXL2 on invasion, metastasis and 67LR expression was evaluated in cholangiocarcinoma cells by shRNA or expressed-plasmid transfection. RESULTS: Expression of 67LR was recognized in 35.62% cholangiocarcinoma tissue, and none in paracancerous tissues. LOXL2 was positively correlated with expression of 67LR. Expression of 67LR or LOXL2 in cholangiocarcinomas was significantly associated with lymph node metastasis, differentiation and poor overall survival. Cox analysis showed that 67LR can act as an independent prognostic biomarker of prognosis in cholangiocarcinoma patients. Expression of LOXL2 decreased by knockdown of 67LR and increased by overexpression of 67LR in cholangiocarcinoma cells. Knockdown of LOXL2 reduced invasion and metastasis in vitro and in vivo. CONCLUSION: 67LR may regulate the expression of LOXL2 to promote invasion and metastasis in cholangiocarcinoma cells. It could be used as an independent prognostic marker in cholangiocarcinoma patients.
Assuntos
Aminoácido Oxirredutases/análise , Neoplasias dos Ductos Biliares/química , Ductos Biliares Intra-Hepáticos , Biomarcadores Tumorais/análise , Colangiocarcinoma/química , Receptores de Laminina/análise , Proteínas Ribossômicas/análise , Idoso , Aminoácido Oxirredutases/genética , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/genética , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Colangiocarcinoma/genética , Colangiocarcinoma/secundário , Feminino , Técnicas de Silenciamento de Genes , Humanos , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Receptores de Laminina/genética , Proteínas Ribossômicas/genética , Taxa de Sobrevida , Regulação para CimaRESUMO
Lysyl oxidase-like 2 (LOXL2) is associated with invasiveness and metastasis in breast cancer. We analyzed the prognostic impact of LOXL2 for breast cancer patients and investigated the role of LOXL2 in breast cancer cell lines. Immunohistochemical study of LOXL2 expression was done in samples from 309 patients. Survival analysis was performed using log-rank test and Cox regression hazard model. After identification of LOXL2 expression in breast cancer cell lines, we performed matrigel invasion and wound-healing assays with LOXL2-silenced cell lines. In the human study, LOXL2 was expressed in 16.2 % of patients. Comparing the LOXL2-positive versus negative groups, there was a significantly higher proportion of estrogen receptor-negative patients (54.0 vs. 37.0 %, respectively; p = 0.029) and triple-negative patients (34.0 vs. 18.0 %; p = 0.022) in the positive group. In multivariate analysis for overall survival and metastasis-free survival, positive LOXL2 was demonstrated as a poor prognostic factor (HR 2.27 and 2.10, respectively). In vitro study indicated that LOXL2 silencing induces a mesenchymal-epithelial transition-like process in basal cell lines (MDA-MB-231 and BT549) associated with decreased invasive and migratory properties. These clinical and preclinical data confirm that higher LOXL2 expression is associated with invasiveness of basal-like breast cancer cells and lower survival of breast cancer patients. Our results suggest the clinical value of LOXL2 as a therapeutic target in breast cancer.
Assuntos
Aminoácido Oxirredutases/análise , Neoplasias da Mama/química , Carcinoma/química , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/análise , Adulto , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/mortalidade , Carcinoma/patologia , Carcinoma in Situ/química , Carcinoma in Situ/genética , Carcinoma in Situ/mortalidade , Carcinoma in Situ/patologia , Linhagem Celular Tumoral , Movimento Celular , Colágeno , Intervalo Livre de Doença , Combinação de Medicamentos , Transição Epitelial-Mesenquimal , Feminino , Humanos , Hibridização In Situ , Estimativa de Kaplan-Meier , Laminina , Pessoa de Meia-Idade , Terapia Neoadjuvante , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Primárias Múltiplas/química , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/mortalidade , Neoplasias Primárias Múltiplas/patologia , Tumor Filoide/química , Tumor Filoide/genética , Tumor Filoide/mortalidade , Tumor Filoide/patologia , Prognóstico , Modelos de Riscos Proporcionais , Proteoglicanas , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Análise de Sobrevida , Análise Serial de Tecidos , Neoplasias de Mama Triplo Negativas/química , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Pseudoexfoliation (PEX) syndrome is an age-related systemic disease of the extracellular matrix, characterized by the presence of amyloid-like fibrillar deposits on the anterior lens capsule. The pathological deposits (PEX material) can obstruct aqueous outflow leading to increased intraocular pressure that in turn can result in glaucoma. PEX syndrome is the most common risk factor for glaucoma. In our previous work, we reported a protocol for the analysis of human lens capsules by MALDI MS imaging. Here, we extend our previous work applying the developed protocol to the analysis of human lens capsules affected by PEX syndrome. We focus our investigation on known components of the PEX material, namely lysyl oxidase-like 1 (LOXL1) and apolipoprotein E (APOE). Our results show that LOXL1 is more abundant in the deposits in the iris region and, alternatively APOE is concentrated in the PEX material accumulated in the pupillary area of the anterior lens capsule. Furthermore, we identify potentially relevant post-translational modifications which may have an important role in promoting the cross-linking processes in PEX syndrome and stabilize aggregate structures within the proteinaceous PEX material. BIOLOGICAL SIGNIFICANCE: This paper is about the identification and localization of apolipoprotein E and lysyl oxidase-like 1 in human lens capsules affected by PEX syndrome by MALDI MS imaging. With this study we expand the clinical application of MALDI MSI toward the use of non-sectioned tissue samples analyzed after in situ enzymatic digestion and advance the knowledge regarding a common pathology like PEX syndrome.
Assuntos
Aminoácido Oxirredutases/metabolismo , Apolipoproteínas E/metabolismo , Diagnóstico por Imagem/métodos , Síndrome de Exfoliação , Proteínas do Olho/metabolismo , Cápsula do Cristalino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aminoácido Oxirredutases/análise , Apolipoproteínas E/análise , Síndrome de Exfoliação/metabolismo , Síndrome de Exfoliação/patologia , Proteínas do Olho/análise , Feminino , Humanos , Cápsula do Cristalino/metabolismo , Cápsula do Cristalino/patologia , Masculino , PlantasRESUMO
With increasing age, dynamic tissues such as lungs, blood vessels and skin lose their ability to both deform and recoil, culminating in tissue stiffening. This loss of tissue elasticity, which profoundly impacts tissue function and thus morbidity, may be due not only to changes in the relative abundance of key extracellular matrix proteins within tissues but also to their accumulation of post-translational modifications. Whilst to date attention has focussed primarily on the age-related non-enzymatic formation of advanced glycation end products, the accumulation of pathological enzyme-mediated cross-links may also lead to age-related tissue stiffening. The lysyl oxidase (LOX) family of enzymes are constitutively expressed in adult tissues and are known to drive the catalysis of cross-links in both fibrillar collagens and elastin. Although immunochemical approaches are commonly used to localise the inactive pro-enzyme of LOX, and biochemical methods are employed to quantify activity in homogenised tissue, they do not allow for the in situ localisation of the enzyme. Thus, we have developed a novel assay to both detect and localise LOX enzyme activity in situ. LOX family members are amine oxidases and this assay uses the principle that an amine substrate in the presence of this class of enzyme will be oxidised to an aldehyde and hydrogen peroxide (H2O2). In turn, H2O2, when combined with luminol and horseradish peroxidase, will produce a light-emitting reaction that can be detected by film autoradiography. The development of a technique to localise specific amine oxidase activity in tissue sections may provide crucial additional information on the exact role played by this class of enzymes in mediating age-related tissue stiffening.
Assuntos
Aminoácido Oxirredutases/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Envelhecimento da Pele/fisiologia , Adolescente , Adulto , Idoso , Aminoácido Oxirredutases/análise , Reagentes de Ligações Cruzadas , Elastina/química , Elastina/metabolismo , Proteínas da Matriz Extracelular/química , Colágenos Fibrilares/química , Colágenos Fibrilares/metabolismo , Humanos , Proteína-Lisina 6-Oxidase/análise , Pele/metabolismo , Especificidade por Substrato , Adulto JovemRESUMO
Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To ensure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest SF), reducing agents [dithiothreitol (DTT) and tris(2-carboxyethyl)phophine (TCEP)], and various concentrations of alkylating agent [iodoacetamide (IAM)]. After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2. Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis.
Assuntos
Aminoácido Oxirredutases/análise , Apoproteínas/análise , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Análise de Sequência de Proteína , Transferrina/análise , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Apoproteínas/metabolismo , Glicosilação , Humanos , Espectrometria de Massas , Proteólise , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transferrina/metabolismoRESUMO
We investigated the role of glutamine synthetases (cytosolic GS1 and chloroplast GS2) and glutamate synthases (ferredoxin-GOGAT and NADH-GOGAT) in the inorganic nitrogen assimilation and reassimilation into amino acids between bundle sheath cells and mesophyll cells for the remobilization of amino acids during the early phase of grain filling in Zea mays L. The plants responded to a light/dark cycle at the level of nitrate, ammonium and amino acids in the second leaf, upward from the primary ear, which acted as the source organ. The assimilation of ammonium issued from distinct pathways and amino acid synthesis were evaluated from the diurnal rhythms of the transcripts and the encoded enzyme activities of nitrate reductase, nitrite reductase, GS1, GS2, ferredoxin-GOGAT, NADH-GOGAT, NADH-glutamate dehydrogenase and asparagine synthetase. We discerned the specific role of the isoproteins of ferredoxin and ferredoxin:NADP(+) oxidoreductase in providing ferredoxin-GOGAT with photoreduced or enzymatically reduced ferredoxin as the electron donor. The spatial distribution of ferredoxin-GOGAT supported its role in the nitrogen (re)assimilation and reallocation in bundle sheath cells and mesophyll cells of the source leaf. The diurnal nitrogen recycling within the plants took place via the specific amino acids in the phloem and xylem exudates. Taken together, we conclude that the GS1/ferredoxin-GOGAT cycle is the main pathway of inorganic nitrogen assimilation and recycling into glutamine and glutamate, and preconditions amino acid interconversion and remobilization.
Assuntos
Aminoácidos/metabolismo , Glutamato Sintase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Zea mays/enzimologia , Aminoácido Oxirredutases/análise , Transporte Biológico , Cloroplastos/metabolismo , Transporte de Elétrons , Expressão Gênica , Glutamato Sintase/genética , Glutamato-Amônia Ligase/genética , Ácido Glutâmico/biossíntese , Nitrogênio/metabolismo , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Zea mays/citologia , Zea mays/metabolismoRESUMO
A membrane-based high-throughput screening (HTS) assay for active D-amino acid oxidase (DAAO) in liquid samples as well as in intact Escherichia coli cells has been developed and optimized. The detection limit of the assay was less than 1 ng per sample. The method proposed can be used for quantitative DAAO determination in the range of 0.13 to 3.60 ng enzyme per probe. The protocol was successfully tested to screen a library of E. coli clones containing mutant DAAOs active toward target substrates.
Assuntos
Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/genética , Colódio/química , Combinação de Medicamentos , Escherichia coli/citologia , Escherichia coli/enzimologia , Escherichia coli/genética , Etorfina/química , Membranas Artificiais , Metotrimeprazina/química , Mutagênese , Mutação , Reprodutibilidade dos TestesRESUMO
A mercaptoacetic acid (MAA)-modified cadmium sulfide (CdS) nanoparticle was synthesized in aqueous solution and used as an oligonucleotide label for the electrochemical detection of nopaline synthase (NOS) terminator gene sequence. The carboxyl groups on the surface of the CdS nanoparticle can be easily covalently linked with NH2-modified NOS oligonucleotide probe sequences. The target ssDNA sequence was fixed onto the electrode surface by covalently linking to a mercaptoethanol self-assembled gold electrode, and the DNA hybridization of target ssDNA with probe ssDNA was accomplished on the electrode surface. The CdS nanoparticles anchored on the hybrids were dissolved in the solution by the oxidation with HNO3 and further detected by a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used for monitoring the hybridization, and the NOS target sequence was satisfactorily detected in the approximate range from 8.0 x 10(-12) to 4.0 x 10(-9) mol L(-1) with a detection limit of 2.75 x 10(-12) mol L(-1) (3sigma). The established method extended the nanoparticle-labeled electrochemical DNA analysis to genetically modified organisms (GMOs) specific sequence samples with higher sensitivity and selectivity.
Assuntos
Aminoácido Oxirredutases/análise , Compostos de Cádmio , Sondas de DNA , Nanopartículas , Oligonucleotídeos/química , Sulfetos , Regiões Terminadoras Genéticas , Agrobacterium tumefaciens/genética , Aminoácido Oxirredutases/genética , Compostos de Cádmio/química , Sondas de DNA/análise , Eletroquímica , Microscopia Eletrônica de Varredura , Nanopartículas/ultraestrutura , Sulfetos/químicaRESUMO
A moderately halophilic, Gram-negative bacterium (strain CG4.1(T)), which was isolated from a solar saltern at Cabo de Gata, a wildlife reserve located in the province of Almería, southern Spain, was subjected to a polyphasic taxonomic study. This organism was an aerobic, motile rod that produced colonies with a yellow pigment. Strain CG4.1(T) grew at salinities of 3-25 % (w/v), at 15-45 degrees C and at pH 5-9. The organism reduced nitrate, hydrolysed starch and had phenylalanine deaminase activity. The major fatty acids were C(18 : 1)omega7c, C(16 : 0) and C(19 : 0) cyclo omega8c. The DNA G+C content was 63.6 mol%. On the basis of phenotypic and phylogenetic data, strain CG4.1(T) appears to be a member of the genus Chromohalobacter and clustered closely with Chromohalobacter species, with 95-96 % similarity between their 16S rRNA gene sequences. However, DNA-DNA relatedness between the isolate and the type strains of Chromohalobacter species was low. Therefore, it is proposed that strain CG4.1(T) represents a novel species, Chromohalobacter salarius sp. nov. The type strain is strain CG4.1(T) (=CECT 5903(T)=LMG 23626(T)).
Assuntos
Halomonadaceae/classificação , Halomonadaceae/isolamento & purificação , Água do Mar/microbiologia , Aerobiose , Aminoácido Oxirredutases/análise , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Genes de RNAr , Halomonadaceae/genética , Halomonadaceae/fisiologia , Concentração de Íons de Hidrogênio , Locomoção , Dados de Sequência Molecular , Nitratos/metabolismo , Hibridização de Ácido Nucleico , Filogenia , Pigmentos Biológicos/biossíntese , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Espanha , Amido/metabolismo , TemperaturaRESUMO
We find that an expression system widely used to chemically induce transgenes of interest in tobacco (Nicotiana tabacum Petit Havana SR1) can cause severe growth defects in this species. This gene expression system has been shown to cause non-specific effects (including growth retardation) in other plant species, but has until now been largely accepted to be a relatively problem-free system for use in tobacco. The expression system is based on the ability of the glucocorticoid dexamethasone (DEX) to activate a non-plant chimeric transcription factor (GVG), which then activates expression of a transgene of interest. The aberrant growth phenotype only manifests itself after DEX application and only occurs in plants in which the constitutive levels of GVG expression are higher than average. We found that approximately 30% of all transgenic plants produced showed some level of growth retardation under our standard growth conditions. However, by modulating irradiance levels following DEX application, we also showed that the manifestation and severity of the aberrant phenotype is highly dependent upon growth conditions, highlighting that such conditions are a critical parameter to consider during all stages of using this gene expression system. We also identified an increase in ACC oxidase gene expression as an early, sensitive and robust molecular marker for the aberrant phenotype. This molecular marker should be valuable to investigators wishing to readily identify transgenic plants in which GVG expression levels are beyond a threshold that begins to produce non-specific effects of the gene expression system under a defined set of growth conditions.