Abstract. Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [~25I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pls 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = r~ 60 >( 10 -9 M) both monomeric and polymeric albumin: the binding is saturable at ~80 nM concentration and 50% inhibition is reached at 5.5 I.tg/ml albumin concentration. Sulfhydryl-reducing agents 13-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.

Biological aging is associated with an increased incidence of cerebrovascular disease. Recent findings indicate that oxidative stress promoting age-related changes of cerebral circulation are involved in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease. The aim of this study was to evaluate the contribution of cerebral microvessels to the oxidative stress during brain aging, by: (i) assessment of precursors for advanced glycation end products (AGE) formation, (ii) activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR), and (iii) the activities of metalloproteinases (MMPs), MMP-2 and MMP-9, involved in synaptogenesis and memory consolidation. The experiments were performed on two groups of male Wistar rats: 15 young (3-6 months old) and 15 aged (18-24 months old) animals. The cerebral microvessels were isolated by mechanical homogenization, the concentration of protein carbonyls and the activity of antioxidant enzymes were evaluated by spectrophotometry, and gelatin SDS-PAGE zymography was employed to evaluate MMP-2 and MMP-9 activities. The results showed that, by comparison with young rats, aged brain microvessels contain: (i) approximately 106 % increase of protein carbonyls production; (ii) approximately 68% higher GPx activity, unmodified activities of SOD and GR; (iii) approximately 30% diminishment in MMP-2 activity, and the specific occurrence of MMP-9 enzyme. The data suggest that the age-related changes of microvessels could increase the propensity for cerebral diseases and might represent, at least in part, a prerequisite for the deterioration of mental and physical status in the elderly.

The aim of the study was to evidence replicative senescence‐induced changes in human amniocytes via flow cytometry, quantitative reverse‐transcription‐polymerase chain reaction (qRT‐PCR) and automated/manual patch‐clamp. Both cryopreserved and senescent amniocytes cultured in BIO‐AMF‐2 medium featured high percentages of pluripotency cell surface antigens SSEA‐1, SSEA‐4, TRA1‐60, TRA1‐81 (assessed by flow cytometry) and expression of pluripotency markers Oct4 (Pou5f1) and Nanog (by qRT‐PCR). We demonstrated in senescent vs cryopreserved amniocytes decreases in mesenchymal stem cell surface markers. Senescence‐associated β‐galactosidase stained only senescent amniocytes, and they showed no deoxyuridine incorporation. The gene expression profile revealed a secretory phenotype of senescent amniocytes (increased interleukin (IL)‐1α, IL‐6, IL‐8, transforming growth factor β, nuclear factor κB p65 expression), increases for cell cycle‐regulating genes (p16INK4A), cytoskeletal elements (β‐actin); HMGB1, c‐Myc, Bcl‐2 showed reduced changes and p21, MDM2 decreased. Via patch‐clamp we identified five ion current components: outward rectifier K+ current, an inactivatable component, big conductance Ca2+‐dependent K+ channels (BK) current fluctuations, Na+ current, and inward rectifier K+ current. Iberiotoxin 100 nmol/L blocked 71% of BK fluctuations, and lidocaine 200 μmol/L exerted use‐dependent Na+ current block. Transient receptor potential (TRP)M7‐like current density at −120 mV was significantly increased in senescent amniocytes. The proinflammatory profile acquired by senescent amniocytes in vitro may prevent their use in clinical therapies for immunosuppression, antiapoptotic and healing effects.

Matrix metalloproteinases play a major role in the process of angiogenesis, an important feature of diabetes complications, cancer or rheumatoid arthritis. High glucose concentrations were reported to augment metalloproteinase‐2 secretion in some cell types. In the present study we investigated the influence of acetylsalicylic acid on metalloproteinase‐2 secretion and expression in endothelial cells cultured for one week in high glucose conditions (25 mM and 33 mM). Metalloproteinase‐2 activity was evidenced by gel zymography, the protein was identified by Western blotting, and the gene expression was quantitated by RT‐PCR. The results indicated a marked inhibitory effect of acetylsalicylic acid at gene expression level (˜43%) and also at secretion level in samples of conditioned media (˜30%) and cellular homogenates (˜70%). This may suggest that acetylsalicylic acid could have a beneficial effect in preventing the angiogenic process that appears in diabetes complications.

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells that are found within mammalian tissues. It is composed of three major classes of biomolecules: (i) structural proteins: collagen and elastin, (ii) specialized proteins: e.g. fibrillin, fibronectin, and laminin, and (iii) proteoglycans.Matrix metalloproteinases (MMPs) are an important class of calcium dependent zinc-containing endopeptidases, involved in the breakdown of ECM, thus playing important roles in growth, remodeling (such as wound healing), and reproduction, among other physiological processes [9]. AbstractDue to their involvement in many pathological conditions, matrix metalloproteinases (MMPs), are very attractive therapeutic targets. Our study focuses on one of them, MMP-2, which is involved in tumor progression and metastasis. Recently, the solution structure of the catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020) was published by Feng et al. Using the Hanessian group published binding affinity data and the structure published by Feng as a basis, we have built a binding affinity model by targeting the S 2 ' pocket of the enzyme with a set of nine α-N-sulfonylamino hydroxamic acid derivatives. Two binding geometries of each ligand have been generated corresponding to two binding modes denoted A and B, respectively, of which the first one is targeting the S 2 ' pocket and the second one the S 1 pocket. For the binding affinity model developed for mode A the computed activities show a rmsd of 0.583 kcal/mol as compared with the experimental data, and a correlation coefficient r 2 of 0.779, while in the case of the binding mode B a rmsd of 0.834 kcal/mol and correlation coefficient r 2 of 0.500, respectively, were obtained. In conclusion, our data suggest a higher probability for the Phe 76 gated S 2 ' open form pocket to accommodate the substituent α versus the wide solvent exposed S 1 subsite, probability which some research groups could have overlooked due to extensive use in their calculations of non revealing S 2 ' pocket open state crystallographic structures instead of NMR ones.