RESUMO
In recent years, significant progress has been made in the fields of nonlinear and ultrafast optics, offering exciting opportunities for terahertz (THz) science and technology. In this study, we present a novel design of a SSBCD (Solid-State-Biased Coherent Detection) device for the coherent detection of ultra-broadband THz pulses. By increasing the number of cross-fingers, we have effectively enhanced the sensitivity of the SSBCD device. The design of stepped and circular structures has successfully expanded the detectable electric field directions while reducing the dependence on the incident field direction. As a result, we have achieved ultra-broadband detection with a high dynamic range and a wide detection angle. These research findings lay a critical foundation for the integration of solid-state ultra-broadband detection into compact and miniaturized terahertz systems.
RESUMO
In recent years, the clinical guidelines for the diagnosis and treatment of rheumatoid arthritis (RA) have been constantly updated. Among the general principles, it is particularly emphasized that, in order to improve the ratio of treat to target(T2T) of RA, doctors and patients should work together to negotiate the details of the guidelines. Therefore, it is important for patients to further understand the disease and clinical guidelines of RA, and to better cooperate with doctors. This study was based on the most concerned issues of RA patients and international standard procedure of guideline study, we organized the working group and introduce the following 16 recommendations constituting the RA patients' practice guidelines.
Assuntos
Antirreumáticos , Artrite Reumatoide , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/terapia , HumanosRESUMO
Hyperuricemia/gout is a common metabolic disease in China, which is a serious threat to people's health. In clinical practice, the standardization of prevention and diagnosis and the rate of treat-to-target need to be improved. There is still a lack of education for the patients about the understanding of clinical guidelines, the disease knowledge and the importance of cooperating with doctors to carry out diagnosis and treatment. From the most concerned issues of the patients, we established the hyperuricemia/gout patient practice guideline working group with multidisciplinary physicians and patients. Seventeen opinions, as the hyperuricemia/gout patient practice guidelines, are proposed in accordance with the relevant principles of the "WHO guidelines development manual" , and with the international normative process, aiming to improve the patients compliance, improve the level of health management of the disease.
Assuntos
Gota , Hiperuricemia , China , Gota/diagnóstico , Gota/terapia , Humanos , Hiperuricemia/diagnóstico , Hiperuricemia/terapia , Guias de Prática Clínica como AssuntoRESUMO
OBJECTIVE: Cervical cancer has become the fourth most common cancer in developing countries. This study aimed to investigate anti-tumor effects of Metformin combining with carboplatin in cervical cell line, HeLa cell. MATERIALS AND METHODS: Human cervical cancer cell line, HeLa cell, was treated with Metformin (5 mmol/l or 10 mmol/l) or/and carboplatin (25 mg/l or 50 mg/l) at different final concentrations, and divided into 8 groups. 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cell viability. Acridine orange/ethidium bromide (AO/EB) staining was used to examine nuclear fragments and cell apoptosis. Annexin V/propidium iodide (PI) staining was employed to detect apoptosis of HeLa cells. Mitochondrial membrane potential of the HeLa cells was evaluated by staining with 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) reagent. RESULTS: MTT results showed that Metformin combining carboplatin significantly reduced HeLa cell viability compared to that of no-drug treatment group (p<0.05). Metformin combining carboplatin significantly increased the amounts of nuclear fragments compared to that of no-drug treatment group (p<0.05). The flow cytometry assay results indicated that Metformin combining carboplatin significantly enhanced the apoptotic rates compared to that of no-drug treatment group (p<0.05). The JC-1 staining findings illustrated that Metformin combining carboplatin significantly decreased the mitochondrial membrane potential compared to that of no-drug treatment group (p<0.05). CONCLUSIONS: Metformin enhanced the inhibitive effects of carboplatin on HeLa cell proliferation. Metformin increased the sensitivity of HeLa cell to the treatment of Carboplatin by activating mitochondrial-associated apoptosis signaling pathway.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Carboplatina/farmacologia , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Mitocôndrias/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Sinergismo Farmacológico , Feminino , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Transdução de Sinais , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
OBJECTIVE: Dopamine receptor (DR) signaling is involved in the pathogenesis of autoimmune diseases. We aimed to measure the expression levels of DR1-5 on B cells from patients with rheumatoid arthritis (RA) and to analyze the relationship between DRs and clinical manifestations, inflammatory biomarkers, functional status and disease activity. METHODS: A total of 29 patients with RA, 12 healthy donors and 12 patients with osteoarthritis (OA) were recruited in this study. Flow cytometry was used to measure the levels of DR1-5 expressed on B cells. The relationships between B cell DR expressions and clinical features in RA patients were analyzed using the Spearman correlation test. RESULTS: The expression levels of B cell DR1-5 in both the RA and OA groups were lower than those in healthy controls. After 3 months of medication, all five receptors were elevated in RA patients, with DR2 and DR3 being significantly increased from the baseline. DR2 expression on B cells was negatively correlated with inflammatory biomarkers and disease activity. CONCLUSION: RA patients had lower expression level of DR2 on B cells compared to the healthy controls, and the level of DR2 negatively correlated with the disease activity. DR2 and DR3 might be novel predictors of patient responses to disease modifying antirheumatic drug therapy.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos B/metabolismo , Osteoartrite/imunologia , Receptores de Dopamina D2/biossíntese , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Receptores de Dopamina D1/biossíntese , Receptores de Dopamina D3/biossíntese , Receptores de Dopamina D4/biossíntese , Receptores de Dopamina D5/biossínteseRESUMO
Ovarian steroid hormones regulate follicular growth and atresia. This study aims to determine whether key ovarian sterol-regulatory genes are differentially expressed in Hu sheep under different short-term nutritional regimens. Estrus was synchronized using intravaginal progestagen sponges. The ewes were assigned randomly to 3 groups. On d 6 to 12 of their estrous cycle, the control (CON) group received a maintenance diet (1.0×M), the supplemented (SUP) group received 1.5×M, and the restricted (R) group received 0.5×M. On d 7 to 12, blood samples were taken. The sheep were slaughtered at the end of the treatment, and their organs and ovaries were collected. The plasma concentrations of urea (P<0.01), total cholesterol (P<0.01), low-density lipoprotein cholesterol (P<0.01), NEFA (P<0.01), FSH (P<0.05), and estradiol (P<0.05) increased with decreasing dietary intake, whereas plasma triglyceride (P<0.01) and triiodothyronine (T3) concentrations decreased (P<0.05). The ewes in the R group had higher spleen weight and percentage of spleen to BW and lower liver and small intestine weights and percentage of liver/stomach to BW than the SUP group ewes (P<0.05). Nutritional restriction decreased the cytochrome p450 (CYP17A1) and estrogen receptor 1 (ESR1) mRNA expression (P<0.05) and increased the cytochrome p450 aromatase (CYP19A1) mRNA expression (P<0.05) in follicles>2.5 mm. Follicle size affected the mRNA expression of very low density lipoprotein receptor (VLDLR), estrogen receptor 2 (ESR2), FSH receptor (FSHR), CYP17A1, and CYP19A1 (P<0.05). In conclusion, we suggest that a potential mechanism by which short-term negative energy balance inhibits follicular growth may involve responses to disrupted reproductive hormone concentrations and influenced the intrafollicular expression of CYP17A1, CYP19A1, and ESR1. This result may be due to increased plasma urea and lipid concentrations.
Assuntos
Regulação da Expressão Gênica/fisiologia , Hormônios Esteroides Gonadais/biossíntese , Lipídeos/sangue , Estado Nutricional , RNA Mensageiro/metabolismo , Ovinos/genética , Ração Animal , Fenômenos Fisiológicos da Nutrição Animal , Animais , Peso Corporal , Dieta/veterinária , Sincronização do Estro , Feminino , Fase Luteal/fisiologia , Ovário/fisiologia , Progestinas/administração & dosagem , Progestinas/farmacologia , RNA Mensageiro/genética , Ovinos/sangue , Ovinos/metabolismo , Ureia/sangueRESUMO
OBJECTIVES: We aimed to investigate the possible effects of vimentin (Vim) and citrullinated Vim (cVim) on proliferation capacity, pro-inflammatory cytokine secretion, and the expression of peptidylarginine deiminase type 4 (PADI4) and receptor activator of nuclear factor kappa B ligand (RANKL) in cultured fibroblast-like synoviocytes (FLSs) from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. METHOD: Human native Vim was citrullinated with rabbit PAD in vitro and detected using a Western blot assay with anti-modified citrulline antibody (anti-MC Ab). FLSs from RA or OA synovial samples were stimulated with Vim or cVim. Cell proliferation capacity was determined using the Celltiter 96 AQueous cell proliferation assay. The concentrations of tumour necrosis factor (TNF)-α, interleukin (IL)-1, and IL-17 were measured by enzyme-linked immunosorbent assay (ELISA). The expression of PADI4 and RANKL was measured by real-time polymerase chain reaction (RT-PCR) and a Western blot assay. RESULTS: Our Western blot assay with anti-MC Ab indicated that the amount of cVim increased significantly after Vim had been incubated with rabbit PAD in vitro. The proliferation capacity and secretion of TNF-α and IL-1 were significantly enhanced in the FLSs of RA patients when treated with cVim. However, when treated with Vim, an inhibitory effect on the proliferation capacity was noted in the FLSs from RA and also from OA patients. cVim significantly increased the expression of PADI4 and RANKL in the FLSs from RA patients. CONCLUSION: cVim seems to have remarkable biological effects on RA as confirmed by the stimulation of proliferation capacity, pro-inflammatory cytokine secretion, and PADI4 and RANKL expression in the FLSs of RA patients.
Assuntos
Artrite Reumatoide/metabolismo , Proliferação de Células/efeitos dos fármacos , Hidrolases/metabolismo , Ligante RANK/metabolismo , Membrana Sinovial/efeitos dos fármacos , Vimentina/farmacologia , Idoso , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1/metabolismo , Interleucina-17/metabolismo , Masculino , Pessoa de Meia-Idade , Osteoartrite/imunologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Aquagenic syringeal acrokeratoderma (ASA) is a rare acquired disorder that develops predominantly in young women. It is clinically characterized by a burning sensation and whitish discolouration on the hands and rarely on the soles after brief immersion in water, which resolves within a short time after drying. Topical aluminium chloride and salicylic acid are reportedly beneficial in some cases. In total, 20 female and 8 male patients with ASA have been reported previously. We present another male patient, who failed to respond to treatment with antihistamines and topical steroids, but responded well to formalin 3% in alcohol without any side-effects.
Assuntos
Formaldeído/administração & dosagem , Hiperidrose/patologia , Imersão/efeitos adversos , Ceratodermia Palmar e Plantar/patologia , Adulto , Humanos , Hiperidrose/complicações , Hiperidrose/tratamento farmacológico , Ceratodermia Palmar e Plantar/tratamento farmacológico , Ceratodermia Palmar e Plantar/etiologia , Masculino , ÁguaRESUMO
Expression patterns of chitinase transcripts induced by N-acetylchitooligosaccharide elicitor were analyzed by northern blot hybridization in order to reveal a signal transduction pathway leading to the activation of class I chitinase genes (Cht-1 and Cht-3), which may play an important role in producing N-acetylchitooligosaccharide elicitor. The transcription level of both genes was enhanced in response to N-acetylchitooligosaccharides larger than pentaose at subnanomolar concentrations. These structure and dose dependencies were consistent not only with those for a 75 kDa high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane, but also with other series of cellular responses including phytoalexin production and the expression of elicitor-responsive genes (EL2, EL3). Therefore, the elicitor signal to evoke these cellular responses including the activation of the chitinase genes could be common and transmitted into cells through the 75 kDa protein. However, the signal transduction pathway for the activation of the chitinase gene appeared to diverge from those for the other elicitor-responsive genes shortly after the signal perception. It was shown that the induction of chitinase expression by N-acetylchitooligosaccharide would require protein phosphorylation, but not de novo protein synthesis. The oxidative burst was demonstrated not to be necessary for transcriptional induction of the all four elicitor-responsive genes (Cht, PAL, EL2, EL3) by N-acetylchitooligosaccharide.
Assuntos
Quitinases/genética , Oligossacarídeos/farmacologia , Oryza/efeitos dos fármacos , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Antracenos/farmacologia , Células Cultivadas , Quitina/farmacologia , Canais de Cloreto/antagonistas & inibidores , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Cinética , NADPH Oxidases/antagonistas & inibidores , Oligossacarídeos/química , Oniocompostos/farmacologia , Oryza/enzimologia , Oryza/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The cytochrome bo3 ubiquinol oxidase contains at least one and possibly two binding sites for ubiquinol/ubiquinone. Previous studies used the photoreactive affinity label 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), a substrate analogue, to demonstrate that subunit II contributes to at least one of the quinol binding sites. In the current work, mass spectroscopy is used to identify a peptide within subunit II that is photolabeled by the azido-Q. Purified cytochrome bo3 was photolabeled as previously described using azido-Q that was not tritiated (i.e., not radiolabeled). Subunit II was then isolated from an SDS-PAGE gel and proteolyzed in situ with trypsin. The resulting peptides were eluted from the gel and then identified using matrix-assisted laser desorption ionization mass spectrometry. The resulting mass spectrum was compared to that obtained by analysis of subunit II that had not been exposed to the photolabel. Using the amino acid sequence, each peak in the mass spectrum of the unlabeled subunit II could be assigned to an expected trypsin fragment. Two additional peaks were observed in the mass spectrum of the photolabeled subunit with m/z 1931.9 and 2287.7. Subtraction of the mass of azido-Q from the peak at m/z 1931.9 results in a mass equivalent to that of a peptide consisting of amino acids 165-178. The assignment of the peak at m/z 2287.7 cannot be made unequivocally and may correspond either to the covalent attachment of azido-Q to peptide 254-270 or to a peptide resulting from incomplete proteolysis. The labeled peptide, 165-178, is within the water-soluble domain of subunit II, whose X-ray structure is known. This peptide is located near the site where CuA is located in the homologous cytochrome c oxidases and can be placed near the interface between subunits I and II.
Assuntos
Citocromos/metabolismo , Escherichia coli/enzimologia , Ubiquinona/análogos & derivados , Sítios de Ligação , Grupo dos Citocromos b , Citocromos/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli , Hidrólise , Modelos Moleculares , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estereoisomerismo , Tripsina , Ubiquinona/química , Ubiquinona/metabolismoRESUMO
By using subtracted probes, two cDNA clones of rice, EL2 and EL3, were isolated as genes responsive within 6 min to N-acetylchitoheptaose, a potent biotic elicitor for phytoalexin biosynthesis. Analyses of the sequence of the cDNAs showed that both of EL2 and EL3 encoded basic proteins with no significant similarities to those of known genes.
Assuntos
Genes de Plantas , Oryza/genética , Extratos Vegetais/biossíntese , Sequência de Aminoácidos , DNA Complementar/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Oligossacarídeos/farmacologia , Oryza/efeitos dos fármacos , Oryza/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sesquiterpenos , Terpenos , FitoalexinasRESUMO
An azidoubiquinone derivative, 3-azido-2-methyl-5-methoxy [3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone-protein interaction and to identify ubiquinone-binding proteins in bovine heart mitochondrial succinate-ubiquinone reductase. When the reductase was incubated with [3H]azido-Q and illuminated with long wavelength UV light, the decrease in the enzymatic activity correlated with the amount of azido-Q incorporated into the protein. When the illuminated, [3H]azido-Q-treated reductase was extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found primarily in the QPs1 subunit. The [3H]azido-Q-labeled QPs1 was purified from labeled reductase by a procedure involving ammonium sulfate fractionation, dialysis, organic solvent extraction, lyophilization, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cold acetone precipitation. The purified, [3H]azido-Q-labeled QPs1 protein was subjected to reductive carboxymethylation prior to digestion by trypsin. One azido-Q-linked peptide, with a retention time of 66.9 min, was obtained by high performance liquid chromatographic separation. The partial amino-terminal sequence of this peptide is GLTISQL-, indicating that this tryptic peptide comprises amino acid residues 113-140 of the revised amino acid sequence of QPs1. The Q-binding domain, using the proposed structure of QPs1, is probably located in the stretch connecting transmembrane helices 2 and 3 that extrude from the surface of the M side of the inner membrane.
Assuntos
Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Conformação Proteica , Succinato Desidrogenase/química , Succinato Desidrogenase/metabolismo , Ubiquinona/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Azidas/metabolismo , Sítios de Ligação , Bovinos , Complexo II de Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Cinética , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/isolamento & purificação , Miocárdio/enzimologia , Oxirredutases/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Especificidade por Substrato , Succinato Desidrogenase/isolamento & purificação , Trítio , Ubiquinona/análogos & derivadosRESUMO
For the investigation of the protein-ubiquinone interaction in the succinate-cytochrome c reductase region of the bovine heart mitochondrial electron transport chain, a series of 5-alkyl-substituted ubiquinone derivatives (5-R-Q0C10) were synthesized and characterized. Syntheses of 5-ethyl-Q0C10, 5-propyl-Q0C10, 5-isopropyl-Q0C10, and 5-butyl-Q0C10, were archived through radical coupling reactions between 2,3-dimethoxy-6-decyl-1,4-benzoquinone (5-H-Q0C10) and the corresponding alkanoyl peroxides. Although the spectral and redox properties of 5-R-Q0C10 are very similar to those of 5-methyl-2,3 dimethoxy-6-decyl-1,4-benzoquinone, the biological electron transfer efficiencies of these derivatives differ significantly. The reducibility of these derivatives by succinate, as measured with succinate-Q reductase and the oxidizability as measured by ubiquinol-cytochrome c reductase, decreased as the size of the substituents increased. 5-Ethyl-Q0C10 has about 50% of the activity of 5-methyl-2,3-dimethoxy-6-decyl-1,4-benzoquinone, whereas molecules with 5-alkyl groups of three or more carbon atoms are virtually inactive as electron acceptors for succinate-Q reductase. Reduced form of the derivative with no substituent at the 5-position, 5-H derivative is more effectively oxidized by ubiquinol-cytochrome c reductase than does the 5-methyl derivative, the native form. The oxidation of 5-H derivative is in a concentration-dependent manner at low concentrations but exhibits a substrate inhibition at higher concentrations. No such substrate inhibition is observed when other 5-substituted Q derivatives are used. 5-H derivative is a better electron acceptor for succinate-Q reductase than any other Q derivatives and does not show substrate inhibition, even at high concentrations. These results indicate that the binding environment of the benzoquinone ring in succinate-Q reductase is more specific than that of ubiquinol-cytochrome c reductase.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Succinato Citocromo c Oxirredutase/metabolismo , Succinato Desidrogenase/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/síntese química , Alquilação , Complexo II de Transporte de Elétrons , Indicadores e Reagentes , Cinética , Oxirredução , Relação Estrutura-Atividade , Ubiquinona/metabolismo , Ubiquinona/farmacologiaRESUMO
For investigation of the protein-ubiquinone interaction in the succinate-cytochrome c reductase region of the bovine heart mitochondrial electron-transport chain, ethoxy-substituted ubiquinone derivatives, 2-ethoxy-3-methoxy- or 3-ethoxy-2-methoxy-5-methyl-6-decyl-1,4-benzoquinone (EtOQ0C10) and 2,3-diethoxy-5-methyl-6-decyl-1,4-benzoquinone [(EtO)2Q0C10], were synthesized and characterized. These compounds were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (Q0C10) by reaction with sodium ethoxide/ethanol in hexane under anaerobic conditions. The products, EtOQ0C10 and (ETO)2Q0C10, were separated by thin-layer chromatography using hexane/ether (3.5:1) as the developing solvent. The Rf values for diethoxy and monoethoxy derivatives are 0.7 and 0.6, respectively. The spectral and redox properties of EtOQ0C10 and (ETO)2Q0C10 are very similar to those of Q0C10. The reducibility of these derivatives by succinate was measured with succinate-Q reductase (SQR), and their oxidizability was measured by ubiquinol-cytochrome c reductase (QCR). Ethoxy ubiquinone derivatives exhibit concentration-dependent inhibition of SQR activity, with (ETO)2Q0C10 being the more potent inhibitor. These derivatives do not inhibit QCR and are reduced by succinate-cytochrome c reductase in an antimycin-insensitive manner. When used as substrate for QCR, EtOQ0C10H2 has about 55%, and (ETO)2Q0C10H2 about 15%, of the activity of Q0C10H2, but with lower apparent Km values. The low efficiency of these compounds as electron donors is apparently not due to their weak binding to QCR. These results indicate that the binding environment of the benzoquinone ring in succinate-Q reductase is very specific and differs from that in ubiquinol-cytochrome c reductase.
Assuntos
Proteínas Musculares/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Animais , Bovinos , Transporte de Elétrons , Ubiquinona/síntese química , Ubiquinona/químicaRESUMO
Cytochrome b was identified as one of the ubiquinone-binding proteins in bovine heart mitochondrial ubiquinol-cytochrome c reductase by photoaffinity labeling using 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]-octyl)-1,4-benzoquinone ([3H]azido-Q). The [3H]azido-Q-labeled cytochrome b protein was purified to homogeneity from the azido-Q-labeled ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea treatment, calcium phosphate column chromatography, acetone precipitation, decanoyl-N-methylglucamide-cholate extraction, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified cytochrome b protein containing 0.5 mol of azido-Q/mol of protein was subjected to reductive carboxymethylation and succinylation prior to digestion by chymotrypsin. Two azido-Q-linked peptides with retention times of 47.1 and 49.0 min were obtained by high performance liquid chromatographic separation. Partial amino-terminal amino acid sequences of these two peptides were determined to be GATVI- and ALVADL-, indicating that these two chymotryptic peptides are from amino residues 142-155 and 326-336. Monospecific polyclonal antibodies against two synthetic ubiquinone-binding peptides, NH2-G-A-T-V-I-T-N-L-L-S-COOH (P-47) and NH2-W-A-L-V-A-D-L-L-T-L-T-W-I-COOH (P-49), were generated in rabbits and purified. Western blotting and enzyme-linked immunosorbent assays showed that the purified antibodies against P-47 reacted with cytochrome b-containing reductases and purified cytochrome b protein. Antibodies against P-47 inhibited activities of succinate-cytochrome c and ubiquinol-cytochrome c reductases only when they were incubated with phospholipid-depleted reductases prior to the replenishment with phospholipid. No inhibition was observed with incubation with phospholipid-containing reductases, indicating that this peptide involved in ubiquinone binding is buried in a phospholipid environment.
Assuntos
Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Mitocôndrias Cardíacas/metabolismo , Estrutura Secundária de Proteína , Ubiquinona/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Sítios de Ligação , Western Blotting , Bovinos , Cromatografia Líquida de Alta Pressão , Quimotripsina , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Ensaio de Imunoadsorção Enzimática , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ubiquinona/análogos & derivados , Ubiquinona/síntese químicaRESUMO
We have formulated the problem of determining whether there has been an upturn in HIV-1 seroconversion incidence over the first five years of follow-up in the Multicenter AIDS Cohort Study (MACS) as that of locating the minimum of a quadratic regression or examination of two-knot piecewise spline models. Under a quadratic model, we propose a method to obtain a direct estimate and a bootstrap estimate for the location of the temporal turning point (local minimum) for HIV-1 seroconversion incidence and three methods to estimate confidence intervals for the location of the turning point for HIV seroconversion incidence: (1) Wald confidence interval estimate with or without log transformation assuming the asymptotic normality and applying the Delta method; (2) asymmetric confidence intervals using Fieller's Theorem and its modification; and (3) bootstrapping confidence intervals. Inferences for the temporal turning point based on Wald tests for a single regression term in a non-linear regression model were not reliable compared to inferences based on confidence intervals placed on calendar time. We present results using these different methods applied to the MACS data and we obtain power estimates to illustrate the performances of different methods.
Assuntos
Soropositividade para HIV/epidemiologia , HIV-1/imunologia , Modelos Estatísticos , Distribuição de Poisson , Estudos de Coortes , Simulação por Computador , Intervalos de Confiança , Seguimentos , Humanos , Incidência , Masculino , Análise de Regressão , Fatores de TempoRESUMO
To investigate the protein-ubiquinone interaction in the bovine heart mitochondrial succinate-cytochrome c reductase region of the respiratory chain, three fluorine substituted ubiquinone derivatives, 2,3-dimethoxy-6-(9'-fluorodecyl)-1,4-benzoquinone (9FQ), 2-methoxy-5-trifluoromethyl-6-decyl-1,4-benzoquinone (TFQ), and 2-methoxy-5-trifluoromethyl-6-(9'-fluorodecyl)-1,4-benzoquinone (9FTFQ), were synthesized. 9FQ was synthesized by radical coupling of Q0 and bis(10-fluoroundecanoyl)peroxide. The latter was prepared by fluorination of undecylenic acid followed by thionylchloride treatment and peroxidation. TFQ was synthesized from 2,2,2-trifluoro-p-cresol by methylation, nitration, reduction, acetylation, nitration, reduction, oxidation, and radical alkylation. 9FTFQ was prepared by the radical alkylation of 2-methoxy-5-trifluoromethyl-1,4-benzoquinone with bis(10-fluoroundecanoyl)peroxide. All three fluoro-Q derivatives are active (greater than 50% the activity of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) when used as electron acceptors for succinate-ubiquinone reductase. However, only 9FQ is active when used as an electron donor for ubiquinol-cytochrome c reductase or as an electron mediator for succinate-cytochrome c reductase. Both TFQ and 9FTFQ are competitive inhibitors for ubiquinol-cytochrome c reductase. A 19FNMR peak-broadening effect was observed for 9FQ when it was reconstituted with ubiquinone-depleted ubiquinol-cytochrome c reductase. A drastic up-field chemical shift was observed for TFQ when it was reconstituted with ubiquinone-depleted reductase. These results indicate that the binding environments of the benzoquinone ring and the alkyl side chain of the Q molecule are different. The strong up-field chemical shift for TFQ, and lack of significant chemical shift for 9FQ, suggest that the benzoquinone ring is bound near the paramagnetic cytochrome b heme.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Succinato Desidrogenase/metabolismo , Ubiquinona/metabolismo , Animais , Bovinos , Transporte de Elétrons , Complexo II de Transporte de Elétrons , Flúor , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Solubilidade , Relação Estrutura-Atividade , Ubiquinona/análogos & derivados , Ubiquinona/químicaRESUMO
In order to investigate the disorders of carbohydrate metabolism and insulin secretion as well as their correlation in patients with liver cirrhosis, we performed an oral glucose tolerance test on 30 patients with liver cirrhosis proven by history, clinical findings, liver function test, radioisotope liver scanning, ultrasonic examination, gastroscopy, barium esophagogram and liver biopsy, compared with 20 healthy controls. Blood glucose and immunoreactive insulin were determined in both groups at 60 min intervals for 180 min. Results showed marked glucose intolerance with peak value 60 min after glucose load in cirrhotic patients with normal fasting blood glucose. Plasma IRI levels were significantly higher in cirrhotic patients than in normal subjects after glucose load (P less than 0.05), especially 180 min after (P less than 0.01). Twelve of 30 cases (40%) showed an abnormal OGTT curve. Of the 12 cases seven (23.3%) showed a diabetic OGTT curve, five (16.7%) an impaired OGTT curve. While eighteen of 30 cases (60%) showed an abnormal OGIRT curve. Among the 18 cases one (3.3%) presented hypersecretic OGIRT curve, twelve (40.0%) with delayed and prolonged peak, and five (16.7%) with hypersecretic OGIRT curve and delayed, prolonged peak.