Fibrinogen Glycation and Presence of Glucose Impair Fibrin Polymerization-An In Vitro Study of Isolated Fibrinogen and Plasma from Patients with Diabetes Mellitus.

BACKGROUND: Fibrin formation and structure may be affected by a plethora of factors, including both genetic and posttranslational modifications, such as glycation, nitration or acetylation. METHODS: The present study examines the effect of fibrinogen glycation on fibrin polymerization, measured in fibrinogen concentration-standardized plasma of subjects with type 2 diabetes mellitus (T2DM) and in a solution of human fibrinogen exposed to 30 mM glucose for four days. RESULTS: The fibrin polymerization velocity (Vmax) observed in the T2DM plasma (median 0.0056; IQR 0.0049‒0.0061 AU/s) was significantly lower than in non-diabetic plasma (median 0.0063; IQR 0.0058‒0.0071 AU/s) (p < 0.05). Furthermore, significantly lower Vmax was observed for glucose-treated fibrinogen (Vmax 0.046; IQR 0.022‒0.085 AU/s) compared to control protein incubated with a pure vehicle (Vmax 0.053; IQR 0.034‒0.097 AU/s) (p < 0.05). The same tendency was observed in the fibrinogen samples supplemented with 6 mM glucose just before measurements. It is assumed that glucose may affect the ability of fibrinogen to form a stable clot in T2DM subjects, and that this impairment is likely to influences the outcomes of some diagnostic assays. As the example, the impaired clotting ability of glycated fibrinogen may considerably influence the results of the standard Clauss method, routinely used to determine fibrinogen concentration in plasma. The stoichiometric analysis demonstrated that spontaneous glycation at both the sites with high and low glycation potential clearly dominated in T2DM individuals in all fibrinogen chains.

Diabetes and Hyperglycemia Affect Platelet GPIIIa Expression. Effects on Adhesion Potential of Blood Platelets from Diabetic Patients under In Vitro Flow Conditions.

Blood platelets play a crucial role in the early stages of atherosclerosis development. The process is believed to require firm adhesion of platelets to atherosclerosis-prone sites of the artery. However, little evidence exists regarding whether the blood platelets of individuals with pathological conditions associated with atherosclerosis have higher potential for adhesion. This process is to a large extent dependent on receptors present on the platelet membrane. Therefore, the aim of the presented study was to determine whether blood platelets from diabetic patients have higher capacity of adhesion under flow conditions and how diabetes affects one of the crucial platelet receptors involved in the process of adhesion-GPIIIa. The study compares the ability of platelets from non-diabetic and diabetic humans to interact with fibrinogen and von Willebrand factor, two proteins found in abundance on an inflamed endothelium, under flow conditions. The activation and reactivity of the blood platelets were also characterized by flow cytometry. Platelets from diabetic patients did not demonstrate enhanced adhesion to either studied protein, although they presented increased basal activation and responsiveness towards low concentrations of agonists. Platelets from diabetic patients were characterized by lower expression of GPIIIa, most likely due to an enhanced formation of platelet-derived microparticles PMPs, as supported by the observation of elevated concentration of this integrin and of GPIIIa-positive PMPs in plasma. We conclude that altered functionality of blood platelets in diabetes does not increase their adhesive potential. Increased glycation and decrease in the amount of GPIIIa on platelets may be partially responsible for this effect. Therefore, higher frequency of interactions of platelets with the endothelium, which is observed in animal models of diabetes, is caused by other factors. A primary cause may be a dysfunctional vascular wall.

Phenolic cross-links: building and de-constructing the plant cell wall.

Covering: Up to 2019Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2nd generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking.

Degradation of lignin ß-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6.

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the ß-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized ß-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis.

Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.