Gold, Silver and Carbon Nanoparticles Grafted on Activated Polymers for Biomedical Applications.

Organic polymers have been applied successfully in fields such as adhesion, biomaterials, protective coatings, friction and wear, composites, microelectronic devices, and thin-film technology. In general, special surface properties with regard to chemical composition, hydrophilicity, roughness, crystallinity, conductivity, lubricity, and cross-linking density are required for the success of these applications. Polymers very often do not possess the surface properties needed for these applications. For these reasons, surface modification techniques which can transform these inexpensive materials into highly valuable finished products have become an important part of the plastics industry. In case of biomedical polymers is plasma treatment used for enhancing cell adhesion, growth and proliferation and to make them suitable for implants and tissue engineering scaffolds. Nanoparticles fascinated scientists for over a century and are now heavily utilized in chemistry, biology, engineering, and medicine. Nowadays nanoparticles can be synthesized reproducibly, modified with seemingly limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. In recent years, focus has turned to therapeutic possibilities for such materials. Structures, which behave as drug carriers, antimicrobial agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to molecule-based systems, but rather for their new physical and chemical properties, which confer substantive advantages in cellular and medical applications. In this review, we provide insights into immobilization, toxicity and biomedical applications of gold, silver and carbon nanoparticles and discuss their grafting to polymer substrates and the influence on cell-material interactions. The adhesion and the response of cells in contact with the surface play an important role in the cytocompatibility of the implant. It is thus important to understand how cells interact with their environment. The main properties decisive for colonization of a material with cells are surface chemistry, roughness, morphology and polarity, wettability and electrical charge.

Xylanases of anaerobic fungus Anaeromyces mucronatus.

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, alpha-xylosidase, beta-xylosidase, alpha-glucosidase, beta-glucosidase, beta-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular beta-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.

Analysis of fatty acid composition of anaerobic rumen fungi.

The fatty acid (FA) composition of fresh mycelia of anaerobic rumen fungi was determined. The fatty acids methyl esters (FAME) of six strains belonging to four genera (Neocallimastix, Caecomyces, Orpinomyces, Anaeromyces) and one unknown strain were analyzed by gas chromatography. All studied fungi possess the same FAs but differences were found in their relative concentrations. The FA profile of anaerobic fungi comprises carbon chains of length ranging from 12 to 24; the most common fatty acids were stearic (C(18:0)), arachidic (C(20:0)), heneicosanoic (C(21:0)), behenic (C(22:0)), tricosanoic (C(23:0)) and lignoceric (C(24:0)) with relative amount representing >4% of total FA. Significant differences were determined for heptadecanoic, oleic, behenic and tricosanoic acids. Rumen anaerobic fungi can contain very long chain fatty acids; we found unsaturated fatty acids including cis-11-eicosenoic (C(20:1)), cis-11,14-eicosadienoic (C(20:2)), erucic (C(22:1n9)), cis-13,16-docosadienoic (C(22:2)) and nervonic (C(24:1)) acids in very small amounts but their presence seems to be unique for anaerobic fungi.

Characterization of chitinases of polycentric anaerobic rumen fungi.

Chitinolytic systems of anaerobic polycentric rumen fungi of genera Orpinomyces and Anaeromyces were investigated in three crude enzyme fractions - extracellular, cytosolic and cell-wall. Endochitinase was found as a dominant enzyme with highest activity in the cytosolic fraction. Endochitinases of both genera were stable at pH 4.5-7.0 with optimum at 6.5. The Orpinomyces endochitinase was stable up to 50 degrees C with an optimum for enzyme activity at 50 degrees C; similarly, Anaeromyces endochitinase was stable up to 40 degrees C with optimum at 40 degrees C. The most suitable substrate for both endochitinases was fungal cell-wall chitin. Enzyme activities were inhibited by Hg(2+) and Mn(2+), and activated by Mg(2+) and Fe(3+). Both endochitinases were inhibited by 10 mmol/L SDS and activated by iodoacetamide.

Involvement of phospholipases C and D in early response to SAR and ISR inducers in Brassica napus plants.

Phospholipid signaling is an important component in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as in responses to environmental stresses, including pathogen attack. We investigated the involvement of both phospholipase C (PLC, EC 3.1.4.11) and D (PLD, EC 3.1.4.4) in early responses to the treatment of Brassica napus plants with the chemical inducers of systemic acquired resistance (SAR): salicylic acid (SA), benzothiadiazole (BTH), and with the inducer mediating the induced systemic resistance (ISR) pathway, methyl jasmonate (MeJA). Rapid activation (within 0.5-6 h treatment) of the in vitro activity level was found for phosphatidyl inositol 4,5 bisphosphate (PIP2)-specific PLC (PI-PLC) and three enzymatically different forms of PLD: conventional PLDalpha, PIP2-dependent PLD beta/gamma, and oleate-stimulated PLDdelta. The strongest response was found in case of cytosolic PIP2-dependent PLD beta/gamma after BTH treatment. PLDdelta was identified in B. napus leaves and was very rapidly activated after MeJA treatment with the highest degree of activation compared to the other PLD isoforms. Interestingly, an increase in the amount of protein was observed only for PLDgamma and/or delta after ISR induction, but later than the activation occurred. These results show that phospholipases are involved in very early processes leading to systemic responses in plants and that they are most probably initially first activated on post translational level.

Acid sphingomyelinase deficiency. Phenotype variability with prevalence of intermediate phenotype in a series of twenty-five Czech and Slovak patients. A multi-approach study.

A multi-approach study in a series of 25 Czech and Slovak patients with acid sphingomyelinase deficiency revealed a broad phenotypic variability within Niemann-Pick disease types A and B. The clinical manifestation of only 9 patients fulfilled the historical classification: 5 with the rapidly progressive neurovisceral infantile type A and 4 with a slowly progressive visceral type B. Sixteen patients (64%) represented a hitherto scarcely documented 'intermediate type' (IT). Twelve patients showed a protracted neurovisceral course with overt or mild neurological symptoms, three a rapidly progressing fatal visceral affection with rudimentary neurological lesion. One patient died early from a severe visceral disease. The genotype in our patients was represented by 4 frameshift and 14 missense mutations. Six were novel (G166R, R228H, A241V, D251E, D278A, A595fsX601). The Q292K mutation (homoallelic, heteroallelic) was strongly associated with a protracted neurovisceral phenotype (10 of 12 cases). The sphingomyelin loading test in living fibroblasts resulted in total degradation from less than 2% in classical type A to 70-80% in classical type B. In the IT group it ranged from 5% to 49% in a 24 h chase. The liver storage showed three patterns: diffuse, zonal (centrolobular), and discrete submicroscopic. Our series showed a notable variability in both the neurological and visceral lesions as well as in their proportionality and synchrony, and demonstrates a continuum between the historical 'A' and 'B' phenotypes of ASM deficiency. This points to a broad phenotypic potential of ASM deficiency, suggesting the existence of still unknown factors independently controlling the storage level in the visceral and neuronal compartments. This report highlights the important position of the IT in the ASM deficiency phenotype classification. We define IT as a cluster of variants combining clinical features of both the classical types. The protracted neuronopathic variant with overt, borderline or subclinical neurology prevails and is important in view of future enzyme replacement therapy. It appears more common in central Europe. The visceral, rapidly progressing early fatal type has been recognized rarely so far.

Study of phospholipases D and C in maturing and germinating seeds of Brassica napus.

Different forms of phospholipase D (dependent on and independent of the presence of phosphatidylinositol 4,5-bisphosphate, PIP(2)) were identified in maturing and germinating seeds of Brassica napus. Both forms were present in cytosolic and membrane fractions of maturing seeds. PIP(2)-dependent activity increased continuously during seed germination, while PIP(2)-independent activity appeared mostly at the very beginning of seed maturation. PIP(2)-dependent activity was detected mainly in the plasma-membrane fraction. Phosphatidylinositol-specific phospholipase C (PI-PLC) was found only in membrane fractions of both types of developing rape seed tissues. The increasing activities of PLC and PIP(2)-dependent PLD were mainly detected in hypocotyls of seedlings. Some biochemical characteristics of both described enzymes are also presented.

Rapid distinguishing of three African green monkey cell lines: different morphology of chromosomes with nucleolus organizer regions (NORs).

Cell lines established from African green monkey kidney cells have been used over years in virology. It is possible to distinguish them by detailed karyology of banded metaphases or by iso-enzyme studies. Different morphology of NOR-bearing chromosomes in three African green monkey cell lines studied in our laboratory enables us to distinguish them in simple conventionally Giemsa-stained slides. Vero cells have two chromosomes with secondary constrictions with active NORs. One chromosome is a small submetacentric with secondary constriction on the long arm in the vicinity of centromere. The second one is rather metacentric with a smaller secondary constriction. In BGM cells we found two submetacentric chromosomes, one has a large secondary constriction, the other has a smaller secondary constriction. BS-C-1 cells have a large metacentric chromosome with secondary constrictions on both arms. In one investigation an additional large acrocentric chromosome with the secondary constriction on the long arm was found. Comparison with morphology of NOR-bearing chromosomes in a Rhesus monkey cell line is discussed. The method described here is particularly useful for a rapid discrimination of cell lines, produced in our Institute, for virological and diagnostic application in virological laboratories of the hygiene service and in hospitals.