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1.
Br J Dermatol ; 184(6): 1153-1160, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33205400

RESUMO

BACKGROUND: Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. We previously showed that genetic variants within the ST18 gene promoter area confer a sixfold increase in the propensity to develop PV. ST18, a transcription factor, was found to be overexpressed in the epidermis of patients with PV. In addition, it was found to promote autoantibody-mediated abnormal epidermal cell-cell adhesion and secretion of proinflammatory mediators by keratinocytes. OBJECTIVES: To delineate the mechanism through which ST18 contributes to destabilization of cell-cell adhesion. METHODS: We used quantitative reverse-transcriptase polymerase chain reaction, immunofluorescence microscopy, a luciferase reporter system, site-directed mutagenesis, chromatin immunoprecipitation (ChIP) and the dispase dissociation assay. RESULTS: The ChIP and luciferase reporter assays showed that ST18 directly binds and activates the TNF promoter. Accordingly, increased ST18 expression contributes to PV pathogenesis by destabilizing cell-cell adhesion in a tumour necrosis factor (TNF)-α-dependent fashion. In addition, dual immunofluorescence staining showed increased expression of both ST18 and TNF-α in the skin of patients with PV carrying an ST18-associated PV risk variant, which was found to be associated with a more extensive PV phenotype. CONCLUSIONS: Our findings suggest a role for TNF-α in mediating the deleterious effect of increased ST18 expression in PV skin.


Assuntos
Pênfigo , Proteínas Repressoras , Autoanticorpos , Adesão Celular , Desmogleína 3/genética , Humanos , Queratinócitos , Pênfigo/genética , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/genética
2.
Clin Exp Dermatol ; 46(4): 663-668, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33190296

RESUMO

BACKGROUND: Pachyonychia congenita (PC) is a rare autosomal dominant disorder featuring palmoplantar keratoderma, nail dystrophy, oral leucokeratosis, pilosebaceous cysts and natal teeth. PC results from dominant mutations in one of five genes (KRT6A, KRT6B, KRT6C, KRT16, KRT17) encoding keratin proteins. AIM: To delineate the clinical and genetic features of PC in a series of Israeli patients. METHODS: We used direct sequencing of genomic DNA, and also used cDNA sequencing where applicable. RESULTS: We collected clinical information and molecular data in a cohort of Israeli families diagnosed with PC (n = 16). Most of the patients were Ashkenazi Jews and had a family history of PC. The most common clinical findings were painful focal plantar keratoderma (94%) accompanied by nail dystrophy (81%), pilosebaceous cysts (31%) and prenatal/natal teeth (13%). In contrast to the high prevalence of KRT6A mutations in other populations, we found that KRT16 mutations were the most common type among Israeli patients with PC (56%). Most (77%) of the Israeli patients with PC with KRT16 mutation carried the same variant (c.380G>A; p.R127H) and shared the same haplotype around the KRT16 locus, suggestive of a founder effect. CONCLUSION: The data gleaned from this study emphasizes the importance of population-specific tailored diagnostic strategies.


Assuntos
Mutação , Paquioníquia Congênita/epidemiologia , Paquioníquia Congênita/genética , Estudos de Coortes , Feminino , Genética Populacional , Genótipo , Humanos , Israel/epidemiologia , Masculino , Epidemiologia Molecular , Fenótipo
3.
Genet Mol Res ; 13(2): 2458-69, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24782000

RESUMO

Bovine papillomaviruses (BPVs) are recognized as causal agents of benign and malignant tumors in cattle. Thirteen types of BPVs have already been described and classified into 3 distinct genera. Divergences in the nucleotide sequence of the L1 gene are used to identify new viral types through the employment of PCR assays with degenerated primers. In the present study, a method for identifying BPVs based on PCR-RFLP and DNA sequencing allowed the identification of a new putative Deltapapillomavirus, designated JN/3SP (JQ280500.1). The analysis of the L1 gene showed that this strain was most closely related to the BPVs -1, -2, -13 , and OaPV1 (71-73% genetic similarity). In this study, we describe the detection of this new putative Deltapapillomavirus type and verify its phylogenetic position within the genus.


Assuntos
Doenças dos Bovinos/virologia , Deltapapillomavirus/genética , Deltapapillomavirus/isolamento & purificação , Filogenia , Sequência de Aminoácidos , Animais , Bovinos , Doenças dos Bovinos/genética , Deltapapillomavirus/classificação , Deltapapillomavirus/patogenicidade , Análise de Sequência de DNA
5.
ISRN Oncol ; 2013: 910849, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24298391

RESUMO

THE MAJORITY OF MALIGNANT CELLS PRESENT GENETIC INSTABILITY WITH CHROMOSOME NUMBER CHANGES PLUS SEGMENTAL DEFECTS: these changes involve intact chromosomes and breakage-induced alterations. Some pathways of chromosomal instability have been proposed as random breakage, telomere fusion, and centromere fission. Chromosome alterations in tumor cells have been described in animal models and in vitro experiments. One important question is about possible discrepancies between animal models, in vitro studies, and the real events in cancer cells in vivo. Papillomaviruses are relevant agents in oncogenic processes related to action on host genome. Recently, many reports have discussed the presence of virus DNA in peripheral blood, in humans and in animals infected by papillomaviruses. The meaning of this event is of controversy: possible product of apoptosis occurring in cancer cells, metastasized cancer cells, or active DNA sequences circulating in bloodstream. This study compares chromosome aberrations detected in bovine cells, in peripheral blood cells, and in BPV lesion cells: the literature is poor in this type of study. Comparing chromosome aberrations described in the different cells, a common mechanism in their origin, can be suggested. Furthermore blood cells can be evaluated as an effective way of virus transmission.

6.
Biomed Res Int ; 2013: 270898, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23865043

RESUMO

Bovine papillomavirus (BPV) is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil.


Assuntos
Doenças dos Bovinos/virologia , Coinfecção/veterinária , Deltapapillomavirus/genética , Deltapapillomavirus/isolamento & purificação , Infecções por Papillomavirus/veterinária , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição/genética , Animais , Biópsia , Brasil , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/patologia , Coinfecção/genética , Coinfecção/patologia , Coinfecção/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Filogenia , Verrugas/patologia , Verrugas/virologia
8.
J Appl Microbiol ; 110(4): 1032-44, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21276147

RESUMO

AIMS: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. METHODS AND RESULTS: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l(-1) ) and culture (CFU l(-1) ) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l(-1) always exceeded CFU l(-1) , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. CONCLUSIONS: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.


Assuntos
Legionella/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Microbiologia da Água , Legionella/genética , Legionella pneumophila/genética , Legionella pneumophila/isolamento & purificação , Temperatura
9.
J Perinatol ; 31(5): 368-72, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21151008

RESUMO

OBJECTIVE: The objective of this study was to compare alterations in the middle cerebral artery (MCA) pulsatility index (PI) and mean velocity (V (mean)) after laser surgery for twin-twin transfusion syndrome (TTTS). STUDY DESIGN: MCA Doppler studies were conducted 1 day before and after laser surgery for TTTS. The pre- and postoperative mean (standard deviation) of the MCA PI and V (mean) z-scores of the recipient and donor fetuses were calculated and compared. Data were analyzed using paired testing and multivariable linear regression models. RESULTS: A total of 103 patients met the study criteria. Recipients' MCA PI increased from -1.29 (1.20) preoperatively to 0.14 (1.52) postoperatively (P < 0.0001), whereas the donors' PI did not change significantly (-0.31 (1.67) to -0.67 (1.29); P = 0.12). There was no significant difference between preoperative and postoperative MCA V (mean) in donors (0.39 (0.83) and 0.38 (0.93), respectively; P = 0.5048) or recipients (0.60 (0.74) and 0.63 (0.90), respectively; P = 0.5324). CONCLUSIONS: Despite the changes in the MCA PI after laser surgery for TTTS, the MCA V (mean) remained constant. These findings may suggest some autoregulatory capacity in the cerebral vessels of the mid-trimester fetus.


Assuntos
Velocidade do Fluxo Sanguíneo , Artéria Cerebral Média/fisiopatologia , Ultrassonografia Doppler em Cores , Ultrassonografia Pré-Natal/métodos , Feminino , Transfusão Feto-Fetal/fisiopatologia , Transfusão Feto-Fetal/cirurgia , Feto , Homeostase , Humanos , Terapia a Laser , Assistência Perioperatória , Circulação Placentária , Gravidez , Complicações Hematológicas na Gravidez/fisiopatologia , Complicações Hematológicas na Gravidez/cirurgia , Segundo Trimestre da Gravidez
10.
Placenta ; 31(7): 611-4, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20451248

RESUMO

OBJECTIVES: The goal of fetoscopic laser surgery for twin-twin transfusion syndrome (TTTS) is to ablate all placental vascular communications, thereby separating the fetal circulatory systems. We sought to ascertain the frequency and clinical implications of residual vascular communications (RVC) post preferential sequential selective laser photocoagulation of communicating vessels (SQLPCV). STUDY DESIGN: TTTS placentas treated via preferential SQLPCV were examined. Patency of vascular communications was assessed via water and/or milk injections. Cases with intrauterine fetal demise or placental disruption were excluded. Outcomes with and without RVC were compared. RESULTS: One hundred seventy-four TTTS patients were treated during the study period. Dual survival at birth was 76% (133/174). Of the 133 dual survivors, 105 (79%) submitted an intact placenta. Five of these 105 placentas had RVC (4.8%). Comparison of RVC versus non-RVC cases revealed the following: gestational age at delivery 28.7(6.5) vs. 33.4(3.3) weeks (p=0.178); recipient birth weight 1287(1061) vs. 1973(610) grams (p=0.020); donor birth weight 1429(1369) vs. 1653(715) grams (p=0.518); donor central/eccentric placental cord insertion 80% vs. 17% (p=0.006). One case required a second laser surgery to complete the laser ablation; this placenta did not have RVC after delivery. Otherwise there were no cases of persistent TTTS. One of the 5 RVC cases (20%) exhibited neonatal findings consistent with twin anemia-polycythemia sequence (TAPS), while none of the non-RVC cases had TAPS (p=0.005). CONCLUSIONS: The rate of RVC was less than 5% among gestations with dual survivors post preferential SQLPCV treatment for TTTS.


Assuntos
Transfusão Feto-Fetal/cirurgia , Fetoscopia , Terapia a Laser , Placenta/irrigação sanguínea , Feminino , Humanos , Recém-Nascido , Gravidez , Resultado do Tratamento
11.
J Perinatol ; 30(3): 188-91, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19798041

RESUMO

OBJECTIVE: To investigate perioperative changes in fetal heart rate (FHR) associated with sequential vs standard selective laser photocoagulation of communicating vessels for the treatment of twin-twin transfusion syndrome (TTTS). STUDY DESIGN: Women with TTTS were treated with the intent of using the sequential procedure. Those who failed this treatment were categorized as having undergone the standard procedure. Pre- and postoperative FHR of donor and recipient fetuses were analyzed. RESULT: Of 98 women, 35 received the standard technique. A postoperative drop in the mean donor FHR was observed in gestations receiving the standard laser, but not in those receiving the sequential technique. In multivariable models that included operative and gestational characteristics, the use of the sequential treatment was associated with improved stability of the FHR of the donor twin. CONCLUSION: The stability in donor FHR following sequential laser ablation when compared with the standard technique is consistent with improved donor hemodynamics.


Assuntos
Transfusão Feto-Fetal/cirurgia , Frequência Cardíaca Fetal , Fotocoagulação a Laser/métodos , Feminino , Humanos , Gravidez , Segundo Trimestre da Gravidez
12.
Poult Sci ; 88(12): 2628-35, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19903962

RESUMO

Liver X receptor alpha (LXRalpha), also referred to as nuclear receptor subfamily 1, group H, member 3 is a member of the nuclear hormone receptor superfamily, and has recently been shown to act as a master transcription factor governing hepatic lipogenesis in mammals. Liver X receptor alpha directly regulates both the expression of other lipogenic transcription factors and the expression of lipogenic enzymes, thereby enhancing hepatic fatty acid synthesis (FASN). In birds, like in humans, fatty acid synthesis primarily occurs in the liver. Whether LXRalpha is involved in hepatic regulation of lipogenic genes remained to be investigated in this species. Here we show that fatty acid synthase and the expression of other lipogenic genes (sterol regulatory element binding protein 1 and steroyl coenzyme A desaturase 1) are induced in chicken hepatoma cells in response to a pharmacological liver X receptor agonist, T0901317. A detailed analysis of the chicken FASN promoter revealed a functional liver X response element. These data define the chicken FASN gene as a direct target of LXRalpha and further expand the role of LXRalpha as a regulator of lipid metabolism in this species.


Assuntos
Ácido Graxo Sintases/metabolismo , Receptores Nucleares Órfãos/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Galinhas , Ácido Graxo Sintases/genética , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado , Dados de Sequência Molecular , Receptores Nucleares Órfãos/agonistas , Sulfonamidas/farmacologia
13.
J Agric Food Chem ; 56(19): 9269-76, 2008 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-18783242

RESUMO

The structural characteristics of the gum exudate of Acacia senegal (gum arabic) have been investigated by monitoring the composition and physicochemical properties before and after treatment with proteolytic enzyme and various alkaline systems. Molecular mass ( M w) and radius of gyration ( R g) measurements were performed using gel permeation chromatography (GPC) coupled to refractive index, UV absorbance, and multiangle light scattering detectors and indicated that the macromolecules present have a compact structure. It was found that treatment with proteolytic enzyme caused the arabinogalactan-protein component (AGP) with average molecular mass approximately 2 x 10 (6) Da to degrade, yielding material of molecular mass approximately 4 x 10 (5) Da, whereas the bulk of the material corresponding to the protein-deficient arabinogalactan component (AG) with molecular mass 4 x 10 (5) remained unaffected. Barium hydroxide was found to hydrolyze the polysaccharide component (AG) itself in addition to the proteinaceous component as demonstrated in control experiments using dextran. However, sodium borohydride/sodium hydroxide treatments were unable to hydrolyze dextran and were assumed to hydrolyze only the proteinaceous component of gum arabic. The AGP component was completely degraded, yielding material of molecular mass approximately 4.5 x 10 (4) Da. It has been concluded, therefore, that the enzyme did not fully hydrolyze all of the protein present and that the AGP component of gum arabic consists of carbohydrate blocks of approximately 4.5 x 10 (4) Da linked to a polypeptide chain consistent with the wattle blossom structure. Because the AGP was degraded to differing extents using a mild and more severe sodium borohydride/sodium hydroxide treatment, it was concluded that the polysaccharide moieties were linked through both O-serine and O-hydroxyproline residues. The gum arabic sample was deglycosylated by treatment with anhydrous hydrogen fluoride and revealed the presence of two putative core proteins of approximately 3 x 10 (4) and approximately 5 x 10 (3) Da, respectively, which correspond to proteins of approximately 250 and 45 amino acids in length. A new model for the structure of the AGP component has been proposed.


Assuntos
Goma Arábica/química , Mucoproteínas/química , Aminoácidos/análise , Compostos de Bário , Glicosilação , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/química
14.
Free Radic Biol Med ; 40(11): 2018-27, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16716902

RESUMO

The reactions of the carbonate and dichloride radical anions, CO3- and Cl2-, with the extracellular matrix glycosaminoglycan hyaluronan (HA) have been studied using the kinetic technique of pulse radiolysis and also by steady-state irradiation combined with gel permeation chromatography/multiangle laser light scattering(gpc/MALLS) to measure the rates of reaction with HA and the yield of HA chain scission, respectively. For comparison, the same measurements were made for the reactions of the free radicals *OH, Br2*-, and N3*. The carbonate and dichloride radical anions were found to react relatively quickly with HA (7.0 x 10(5) and 6.9 x 10(6) dm3 mol(-1) s(-1), respectively) although they are much less reactive than the hydroxyl radical, *OH. Significant yields (20 and 38%, respectively) of chain scission of HA by these radical anions were also determined from the gpc/MALLS experiments, providing some support for their potential participation in the depolymerization of HA in vivo. These results are compared with data obtained for the other free radicals (hydroxyl, azide radicals, and dibromide radical anions) investigated in this study in order to gain an insight into their mechanism of reaction with HA. Earlier chain scission yields of HA by hydroxyl radicals determined by the authors have also been revised using the gpc/MALLS technique employed in the current study. The yields of 52% (absence of air) and 44% (in air) are much lower than the previous values. In the current study, the effect of oxygen on the yields of HA chain breaks is discussed in terms of the reactivity of HA peroxyl radicals in the presence of superoxide radical anions. The relevance of the results of this study to mechanisms of inflammation is discussed.


Assuntos
Carbonatos/metabolismo , Cloretos/metabolismo , Ácido Hialurônico/metabolismo , Inflamação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Hialurônico/química , Cinética , Peso Molecular
15.
Kidney Int ; 69(2): 257-65, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16408114

RESUMO

Anecdotal evidence suggests that high fibre supplementation of dietary intake may have health benefits in renal disease related to alterations in circulating levels of short-chain fatty acids. The aim of the study was to examine the hypothesis that dietary manipulation may increase serum butyrate and thus have potential beneficial effects in renal disease. We examined the effect of dietary supplementation with a gum arabic sample of standardized molecular characteristics, Acacia(sen) SUPERGUM EM2 (SUPERGUM), on systemic levels of butyrate in normal human subjects. In an in vitro study, we also examined the potential role of butyrate in modifying the generation of the profibrotic cytokine transforming growth factor-beta (TGF-beta1) by renal epithelial cells. Following 8 weeks of dietary supplementation with 25 g/day of SUPERGUM, there was a two-fold increase in serum butyrate (n=7, P=0.03). In vitro work demonstrated that exposure of renal epithelial cells to elevated concentrations of butyrate suppressed both basal and stimulated TGF-beta1 synthesis. The action of butyrate was mediated by suppression of the extracellular signal-regulated kinase/mitogen-activated protein kinase signalling pathway. In addition, butyrate exposures reduced the response of renal epithelial cells to TGF-beta1 as assessed by luciferase activity of a TGF-beta-responsive reporter construct. Attenuation of TGF-beta1 signalling was associated with reduced phosphorylation of Smad 3 and decreased trafficking of TGF-beta1 receptors into signalling, non-lipid raft-associated membrane fractions. In conclusion, the data demonstrate that dietary supplementation with SUPERGU increased serum butyrate, which at least in vitro has beneficial effects on renal pro-fibrotic cytokine generation.


Assuntos
Butiratos/sangue , Goma Arábica/farmacologia , Rim/efeitos dos fármacos , Fator de Crescimento Transformador beta/biossíntese , Células Cultivadas , Suplementos Nutricionais , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glucose/farmacologia , Humanos , Rim/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1 , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
16.
Poult Sci ; 82(1): 54-61, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12580245

RESUMO

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Galinhas/genética , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Proteínas Estimuladoras de Ligação a CCAAT/química , Cricetinae , Proteínas de Ligação a DNA/química , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Polimorfismo Genético , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fatores de Transcrição/química
17.
Arch Biochem Biophys ; 411(1): 73-82, 2003 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-12590925

RESUMO

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.


Assuntos
Ácido Hialurônico/química , Ácido Peroxinitroso , Cromatografia em Gel , Radicais Livres , Radical Hidroxila , Cinética , Peso Molecular , Soluções
18.
Biopolymers ; 59(4): 191-204, 2001 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-11473346

RESUMO

Using a variety of rheological techniques, the behavior of hyaluronan (M(w) 0.8-2.2 x 10(6)), cross-linked hyaluronan (hylan) (M(w) 1.8-12.5 x 10(6)), and Healon (M(w) approximately 5 x 10(6)) (a proprietary hyaluronan) was studied over a large range of molecular weights. The object was to study the effect of the cross-links in hylan on the various rheological parameters, in comparison with linear hyaluronan. There are significant differences. The Huggins constant and the critical overlap parameter C*[eta] are considerably lower for hylan and an increase in moduli at low frequencies was observed for hylan compared with the hyaluronan samples at all molecular weights studied. The results point to a difference in structure in dilute solution for hylan due to the ability to form networks, which can be removed by pressure filtration. In contrast, we do not find an increase of the steady shear viscosity and elastic modulus at higher concentrations when a homogeneous entangled network is reached. We attribute this behavior to the semirigid character of the hyaluronan chain and to the predominance of entanglements over the cross-link points present in hylan in the semidilute domain. Due to the higher apparent molecular weights that are possible with hylan structures but not with the hyaluronans currently available, a wider range of applications can be achieved with hylans when viscoelasticity is required, particularly for the viscosupplementation of synovial fluid damaged by osteoarthritis.


Assuntos
Ácido Hialurônico/química , Animais , Bactérias/química , Biopolímeros/química , Biopolímeros/isolamento & purificação , Biopolímeros/uso terapêutico , Galinhas , Reagentes de Ligações Cruzadas , Elasticidade , Humanos , Ácido Hialurônico/análogos & derivados , Ácido Hialurônico/isolamento & purificação , Ácido Hialurônico/uso terapêutico , Técnicas In Vitro , Microscopia de Força Atômica , Peso Molecular , Osteoartrite do Joelho/tratamento farmacológico , Reologia , Soluções , Termodinâmica , Viscosidade , Água
19.
Int J Pharm ; 212(1): 73-80, 2001 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-11165822

RESUMO

Release of salicylic acid, diclofenac acid, diclofenac diethylamine and diclofenac sodium, from lyotropic structured systems, namely; neat and middle liquid crystalline phases, across mid-dorsal hairless rat skin into aqueous buffer were studied. Release results were compared with those from the isotropic systems. The donor systems composed of the surfactant polyoxyethylene (20) isohexadecyl ether, HCl buffer of pH 1 or distilled water and the specific drug. High performance liquid chromatography (HPLC) methods were used to monitor the transfer of the drugs across the skin barrier. Results indicated that the rate-determining step in the transport process was the release of the drug from the specified donor system. Further, apparent zero order release was demonstrated with all systems. Except for diclofenac sodium, drug fluxes decreased as the donor medium changed from isotropic to anisotropic. The decrease in fluxes was probably due to the added constrains on the movement of drug molecules. By changing the anisotropic donor medium from neat to middle phase, drug flux decreased in case of salicylic acid and diclofenac sodium. In the mean time, flux increased in case of the diethylamine salt and appeared nearly similar in case of diclofenac acid. Rates of drug transfer across the skin from the anisotropic donors seemed to be largely controlled by the entropy contribution to the transport process. The type and extent of drug-liquid crystal interactions probably influenced the latter.


Assuntos
Anti-Inflamatórios não Esteroides/farmacocinética , Diclofenaco/farmacocinética , Ácido Salicílico/farmacocinética , Pele/metabolismo , Tensoativos/administração & dosagem , Animais , Diclofenaco/administração & dosagem , Masculino , Ratos , Ratos Sprague-Dawley , Ácido Salicílico/administração & dosagem
20.
Int J Biol Macromol ; 27(5): 337-48, 2000 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-10998492

RESUMO

Shear flow, dynamic oscillation and extensional viscosity measurements were used to compare the rheological performance of several hylan samples (M(v) 1.6, 3.2, 3.7, 4.7 and 5.6x10(6)) and hyaluronan (M(v) 1.4 and 1.8x10(6)) before and after hydroxyl radicals (*OH) induced degradation. It was found that the higher molecular weight cross-linked structure of hylan was more resistant to degradation than hyaluronan and that this superior stability was reflected in various rheological parameters. The *OH degradation of the initial hylan and hyaluronan samples produced a range of polysaccharides based on hylan and hyaluronan with molecular weight covering a range from 0.5-5.6x10(6). The rheological parameters associated with the polysaccharides could then also be studied. Zero shear values of the complex viscosity (eta*), dynamic viscosity (eta') and shear viscosity (eta) were calculated using the method of Morris(1) and shown to approach the same value at zero shear or frequency. An adaptation of the method of Gibbs et al. gave a 'master curve' for the storage and loss modulus of hyaluronan and hylan, which encompasses a 10-fold molecular weight and a 5-fold concentration variation. In all instances for hylan, the storage modulus predominates over the loss modulus, whereas for hyaluronan, the reverse is true, demonstrating the greater elasticity of hylan throughout the whole experimental range of molecular weights and concentrations.


Assuntos
Ácido Hialurônico/análogos & derivados , Ácido Hialurônico/química , Radical Hidroxila/química , Animais , Humanos , Radical Hidroxila/farmacologia , Articulações/química , Peso Molecular , Reologia/efeitos dos fármacos
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