Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

Sensitivity of Helicoverpa armigera nucleopolyhedrovirus polyhedra to sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is commonly used to extract polyhedra from infected cells and diseased dead larval tissues. It was found, however, that 80% of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) polyhedra produced via cell culture were damaged after 30 min of 0.5% SDS treatment whereas only 20% of in vivo produced polyhedra were damaged by the same treatment. Transmission and scanning electron microscopy revealed that the damaged polyhedra had lost their polyhedron envelopes and virions were dislodged from the polyhedrin matrix, leaving empty spaces that were previously occupied by the occluded virions. Up to 20% in vitro produced polyhedra were resistant to SDS and remained intact, even after a 24 h exposure to SDS. This sensitivity to SDS was observed across a range of cell culture media, including serum supplemented media. Electron microscopy also revealed that the inferior polyhedron envelope of in vitro produced polyhedra is likely due to poor interaction between the polyhedron envelope, polyhedron envelope protein (PEP), and polyhedrin matrix. The PEP gene was cloned and sequenced and mutations in this gene were ruled out as an explanation. In vitro produced polyhedra that were passed through insect larva once were resistant to SDS, indicating that a critical component is lacking in insect cell culture medium used for producing HaSNPV or the cells growing in culture are inefficient in some ways in relation to production of polyhedra. (C) 2002 Elsevier Science (USA). All rights reserved.

Glucosamine and chondroitin sulfate in the repair of osteochondral defects in dogs - clinical-radiographic analysis

Among the proposed treatments to repair lesions of degenerative joint disease (DJD), chondroprotective nutraceuticals composed by glucosamine and chondroitin sulfate are a non-invasive theraphy with properties that favors the health of the cartilage. Although used in human, it is also available for veterinary use with administration in the form of nutritional supplement independent of prescription, since they have registry only in the Inspection Service, which does not require safety and efficacy testing. The lack of such tests to prove efficacy and safety of veterinary medicines required by the Ministry of Agriculture and the lack of scientific studies proving its benefits raises doubts about the efficiency of the concentrations of such active substances. In this context, the objective of this study was to evaluate the efficacy of a veterinary chondroprotective nutraceutical based on chondroitin sulfate and glucosamine in the repair of osteochondral defects in lateral femoral condyle of 48 dogs, through clinical and radiographic analysis. The animals were divided into treatment group (TG) and control group (CG), so that only the TG received the nutraceutical every 24 hours at the rate recommended by the manufacturer. The results of the four treatment times (15, 30, 60 and 90 days) showed that the chondroprotective nutraceutical, in the rate, formulation and administration at the times used, did not improve clinical signs and radiologically did not influence in the repair process of the defects, since the treated and control groups showed similar radiographic findings at the end of the treatments.

Evaluation of the total antioxidant capacity of flavored water and electrochemical purine damage by sulfate radicals using a purine-based sensor

In this study, a method for the electrochemical quantification of the total antioxidant capacity (TAC) in beverages was developed. The method is based on the oxidative damage to the purine bases, adenine or guanine, that are immobilized on a glassy carbon electrode (GCE) surface. The oxidative lesions on the DNA bases were promoted by the sulfate radical generated by the persulfate/iron(II) system. The presence of antioxidants on the reactive system promoted the protection of the DNA bases immobilized on the GCE by scavenging the sulfate radical. Square-wave voltammetry (SWV) was the electrochemical technique used to perform this study. The efficiencies of five antioxidants (ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol) in scavenging the sulfate radical and, therefore, their ability to protect the purine bases immobilized on the GCE were investigated. These results demonstrated that the purine-based biosensor is suitable for the rapid assessment of the TAC in flavors and flavored water.

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas

Biophysical Chemistry 110 (2004) 83–92

Characterization of the [NiFe] Hydrogenase from the sulfate reducer Desulfovibrio vulgaris Hildenborough

The [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough was isolated from the cytoplasmic membranes and characterized by EPR spectroscopy. It has a total molecular mass of 98.7 kDa (subunits of 66.4 and 32.3 kDa), and contains 1 nickel and 12 Fe atoms per heterodimer. The catalytic activities for hydrogen consumption and production were determined to be 174 and 89 umol H2 min-1 mg -1, respectively. As isolated, under aerobic conditions, this hydrogenase exhibits EPR signals characteristic of the nickel centers in [NiFe] hydrogenases (Ni-A signal at gx,y,z=2.32, 2.23 and ~2.0 and Ni-B signal at gx,y,z=2.33, 2.16 and ~2.0) as well as an intense quasi-isotropic signal centered at g=2.02 due to the oxidized [3Fe-4S] center. The redox profile under hydrogen atmosphere is remarkably similar to that of other [NiFe] hydrogenases. The signals observed for the oxidized state disappear, first being substituted by the Ni-C type signal (gx,y,z=2.19, 2.14, ~2.01), which upon long incubation under hydrogen yields the split Ni-C signal due to interaction with the reduced [4Fe-4S] centers.

Fast-to-fed shift in glucose homeostasis: clues to an earlier detection of human prediabetic states

FCM: UC Bioquímica I - PhD Thesis

The ubiquitin editing enzyme A20 maintains immune homeostasis and prevents autoimmunity

Dissertation presented to obtain the Doctorate degree (Ph.D.) in Biology at Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Characterization of molybdenum and tungsten formate dehydrogenases from sulfate reducing bacteria

Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Formate metabolism in sulfate reducing bacteria

Dissertation presented to obtain the Ph.D degree in Biochemistry

Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Sep 1;65(Pt 9):926-9

Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jul 1;64(Pt 7):593-5

Purification and Preliminary Characterization of Tetraheme Cytochrome and Adenylylsulf te Reductase from the Peptidolytic Sulfate-Reducing Bacterium Desulfovibrio aminophilus DSM 12254

Bioinorganic Chemistry and Applications Volume 3 (2005), Issue 1-2, Pages 81-91

Structural and electron paramagnetic resonance (EPR) studies of mononuclear molybdenum enzymes from sulfate-reducing bacteria

Acc. Chem. Res., 2006, 39 (10), pp 788–796 DOI: 10.1021/ar050104k

Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

J Biol Inorg Chem (2004) 9: 145–151 DOI 10.1007/s00775-003-0506-z