Synthesis and characterization of new biodegradable hyaluronan alkyl derivatives

Several new biocompatible and degradable materials were prepared by chemical modification of sodium hyaluronate. The method of activation of hyaluronate by cyanogen bromide was used and subsequent reaction with nucleophile led to the formation of carbamate. This modification of hydroxyl groups of glycosaminoglycans preserves the carboxyl groups and retains properties of polyelectrolyte. This method affords derivatives easily and the reaction condition correlates with degree of substitution. The experimental results show the effect of reaction conditions (reaction time, ratio of reactants) and effect of substitution on biodegradability. The obtained materials were characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy.     

The combined effect of pycnogenol with ascorbic acid and trolox on the oxidation of lipids and proteins

Pycnogenol (PYC), a procyanidin-rich extract of French maritime pine bark (Pinus pinaster) has strong antioxidant potential and promotes cellular health. The aim of this study was to investigate a possible cooperation of natural antioxidant PYC with synthetic antioxidants ascorbic acid and trolox in the model system of lipid peroxidation determined as conjugated dienes formation in liposomes and on the oxidation of proteins (in BSA and plasma proteins) determined as protein carbonyls. The present study shows that PYC and trolox significantly increased inhibition of lipid peroxidation initiated by copper acetate and tert-butylhydroperoxide in concentration and time dependence compared with untreated unilamellar liposomes. PYC and trolox added simultaneously to the oxidized liposomes exerted an additive preventive effect. PYC s inhibitory effect on formation of carbonyl compounds in BSA and plasma proteins, oxidized by two oxidative systems--H2O2/FeSO4 and HOCl, were studied in co-operation with other synthetic antioxidants--ascorbic acid and trolox. We found the synergistic or additive effect of PYC with mentioned antioxidants.     

Intra- and extraneuronal changes of immunofluorescence staining for TNF-alpha and TNFR1 in the dorsal root ganglia of rat peripheral neuropathic pain models

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-alpha and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury. 2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-alpha and TNFR1 proteins. The TNF-alpha and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-alpha and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-alpha and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury. 3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-alpha and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFalpha IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-alpha after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFalpha IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites. 4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF. 5. Our results suggest more sources of TNF-alpha protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-alpha molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-alpha effect on the primary sensory neurons can be mediated following nerve injury.     

Impact of Ginkgo Biloba Extract EGb 761 on ischemia/reperfusion - induced oxidative stress products formation in rat forebrain

Dysbalance in reactive oxygen/nitrogen species is involved in the pathogenesis of cerebral ischemia/reperfusion injury (IRI). Ginkgo biloba extract (Egb 761) pre-treatment was used to observe potential antioxidant/neuroprotective effect after global ischemia/reperfusion. Egb 761 significantly decreased the level of lipoperoxidation (LPO) in rat forebrain total membrane fraction (homogenate) induced by in vitro oxidative stress (Fe(2+)+H(2)O(2)). In animals subjected to four-vessel global ischemia for 15 min and 2-24 h reperfusion the EGb pretreatment slightly decreased LPO in forebrain homogenate. However, as detected in EGb treated group, the LPO-induced lysine conjugates are attenuated in comparison to non-treated IRI animals. EGb significantly improved parameters which indicate forebrain protein oxidative damage after IRI. The intensity of tryptophane fluorescence was increased by the 18.2% comparing to non-treated IRI group and bityrosine fluorescence was significantly decreased in ischemic (21%) and 24 h reperfused (15.9%) group in comparison non-treated IRI group. In addition, the level of total free SH- groups in pre-treated animals was significantly higher comparing to non-treated animals. Our results indicate that extract of EGb 761 has potent antioxidant activity and could play a role to attenuate the IRI-induced oxidative protein modification and lipoperoxidation in the neuroprotective process.     

Distribution of secretory pathway Ca2+ ATPase (SPCA1) in neuronal and glial cell cultures

1. Secretory pathway Ca(2+) ATPase type 1 (SPCA1) is a newly recognized Ca(2+)/Mn(2+)-transporting pump localized in membranes of the Golgi apparatus. 2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues. 3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains. 4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells. 5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically. 6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, alpha-tubulin or MBP. 7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells. 8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.     

Balancing co-stimulation and inhibition with BTLA and HVEM

The interaction between B- and T-lymphocyte attenuator (BTLA), an inhibitory receptor whose extracellular domain belongs to the immunoglobulin superfamily, and herpesvirus-entry mediator (HVEM), a co-stimulatory tumour-necrosis factor receptor, is unique in that it is the only receptor-ligand interaction that directly bridges these two families of receptors. This interaction has raised many questions about how receptors from two different families could interact and what downstream signalling events might occur as a result of receptor ligation. As we discuss, recent studies show that engagement of HVEM with its endogenous ligand (LIGHT) from the tumour-necrosis factor family induces a powerful immune response, whereas HVEM interactions with BTLA negatively regulate T-cell responses.     

Relationship among urinary albumin excretion rate, lipoprotein lipase PvuII polymorphism and plasma fibrinogen in type 2 diabetic patients

Plasma fibrinogen level represents a strong cardiovascular risk factor and is regulated by an interplay of genetic and environmental factors. Hyperfibrinogenemia frequently occurs in cluster with dyslipidemia within the frame of insulin resistance syndrome (IRS) and type 2 diabetes mellitus. Genetic variants with a pleiotropic effect have been proposed to cause IRS features including hyperfibrinogenemia. We studied the influence of polymorphisms in lipoprotein lipase (LPL) gene, beta-fibrinogen gene (FIBB) and environmental factors on plasma fibrinogen levels in type 2 diabetes patients. 131 type 2 diabetes patients (mean age 62+/-10 years, 33% male) were genotyped for polymorphisms in LPL gene (intron 6 PvuII, intron 8 HindIII) and FIBB gene (-148C/T, -455G/A) by PCR-RFLP method. Fibrinogen was measured by thrombin coagulation method, albuminuria by immunoturbidimetric assay. Polymorphism LPL PvuII showed a gene-dose effect on fibrinogen levels, with the highest fibrinogen in P-P- homozygotes (p = 0.05, analysis of variance). P-carriers (P-P- and P+P- combined) had significantly higher fibrinogen levels compared with P+P+ homozygotes (3.74+/-1.40 g/l vs 3.06+/-1.20 g/l, p=0.03). Other studied polymorphisms were not significantly related to fibrinogen levels. Age- and sex-adjusted fibrinogenemia correlated significantly with albuminuria (r = 0.48, p=0.001), serum uric acid (r = 0.42, p=0.006) and serum creatinine (r = 0.32, p=0.04). Multiple stepwise linear regression identified interaction term of LPL PvuII and albuminuria as an independent predictor of fibrinogen level, explaining 18% of fibrinogen variance. Albuminuria thus appears to be the best predictor of fibrinogen plasma levels in type 2 diabetic patients. Relationship between albuminuria and fibrinogenemia may be modified by the genotype LPL PvuII, which also shows a weak association with plasma fibrinogen level in type 2 diabetes patients.     

Multiblock reducible copolypeptides containing histidine-rich and nuclear localization sequences for gene delivery

Polyplexes sensitive to redox potential gradients represent promising gene delivery vectors. High molecular weight polypeptides containing disulfide bonds in the backbone were synthesized by an oxidative copolymerization of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide. The synthetic approach allowed an easy synthesis of reducible copolypeptides (rCPP) with different relative contents of the HRP and NLS sequences. Cytotoxicity and transfection activity of rCPP-based DNA polyplexes were evaluated in vitro. In comparison with control polyethylenimine (PEI), only minimum toxic effects of rCPPs were observed on the metabolic activity and membrane integrity of human endothelial cells. These findings are predominantly ascribed to favorable structural features like lower charge density and higher chain rigidity of the rCPPs compared to PEI and also to a reductive intracellular and plasma membrane degradation. Transfection activity in all tested cell lines increased with increasing content of the HRP sequence in the rCPPs, while no clearly measurable effect of the NLS sequence was found.     

Classification of weed vegetation of arable land in the Czech Republic and Slovakia

Numerical classification of 2653 geographically stratified relevés of weed vegetation from the Czech and Slovak Republics was performed with cluster analysis. Diagnostic species were determined for each of the seven main clusters using statistical measures of fidelity. The classification reflected clear distinctions between lowland (mostly calcicole) and highland (mostly calcifuge) sites, spring and summer phenological stages, and cereals and root crops. The results of the cluster analysis were compared with traditional phytosociological units. Two clusters corresponded to calcifuge weed vegetation of theScleranthion annui alliance; one cluster represented the vegetation of root crops on moist soils of theOxalidion europaeae alliance; one cluster contained thermophilous weed vegetation of theCaucalidion lappulae alliance; two clusters included weed vegetation of root crops and of stubble fields, which can be assigned to theCaucalidion, Panico-Setarion,Veronico-Euphorbion andEragrostion alliances; one cluster included vernal weed vegetation in little disturbed habitats of theCaucalidion lappulae andScleranthion annui alliances. Our analysis did not support the concept of theSherardion andVeronico-Taraxacion alliances, which were included in earlier overviews of the vegetation units of the Czech Republic and Slovakia.