Comparative neuro tissue engineering using different nerve guide implants.

At the moment autologous nerve grafting remains the only reasonable technique for reconstruction of peripheral nerve defects. Unfortunately, this technique has a lot of complications and disadvantages. These problems are related to the autologous nerve that is harvested for this procedure. Donor site morbidity with loss of sensitivity, painful neuroma formation and of course the restricted availability of autologous nerves stimulates the idea for alternative techniques on that field. In this paper we describe our experience with different graft materials for reconstruction of a 2 cm nerve gap in a median nerve model in rats. After implantation of various materials (biological/synthetic) the main experiments were conducted with a synthetic, biodegradable nerve conduit seeded with autologous Schwann cells. With this material we were able to reconstruct successfully a 2 cm gap in the rat median nerve. Regeneration with this material was found to be equally to an autologous nerve graft.

The presence of microcystins and other cyanobacterial bioactive peptides in aquatic fauna collected from Greek freshwaters.

Toxic bloom-forming cyanobacteria can cause animal death and adversely affect human health. Blooms may contain microcystins (MCs), cyanobacterial heptapeptide hepatotoxins and other peptides such as anabaenopeptins and anabaenopeptilides. MCs have been shown to occur in various aquatic organisms including mussels, water snails, crustaceans and fish. Muscle and viscera samples from eight species of fish (Acipenser gueldenstaedtii, Carassius auratus, Carassius gibelio, Cyprinus carpio, Perca fluviatilis, Rutilus rubilio, Silurus aristotelis and Silurus glanis), a frog (Rana eperotica), a mussel (Anodonta sp.) and a water snail (Viviparus contectus) were analyzed by high-performance liquid chromatography (HPLC), protein phosphatase 1 (PP1) inhibition assay (PP1IA) and ELISA. MC(s) was detected in all fish, frog, mussel and water snail samples tested by PP1IA and ELISA, including the frog R. eperotica and the freshwater snail V. contectus, in which the occurrence of MCs was not previously known. MC concentration ranged from 20 to 1500 ng g(-1)dw and from 25 to 5400 ng g(-1)dw in muscle and visceral tissue of fishes and frogs, respectively. In mussel and water snail tissue MC concentration ranged from 1650 to 3495 ng g(-1)dw. HPLC analysis revealed peaks having the same UV spectrum as anabaenopeptin- or anabaenopeptilide-like compounds, not previously known to occur in aquatic fauna tissue. The concentrations of the compounds detected ranged from 1.5 to 230 microg g(-1)dw. Comparison of the PP1IA and ELISA showed that values obtained with PP1IA where higher than those obtained with ELISA. Anabaenopeptins and/or anabaenopeptilides occurring in faunal tissue may account for the higher PP1IA values as we found that PP1 activity was inhibited by the purified anabaenopeptins A (45-60% inhibition) and B (5-75% inhibition). Purified anabaenopeptilides 90A and 90B exhibited weaker PP1 inhibition activity (5-35 and 5-23% inhibition, respectively). This is the first report of MC occurrence in aquatic animals collected from freshwaters of southern Europe.

Limnothrix redekei (Van Goor) Meffert (Cyanobacteria) strains from Lake Kastoria, Greece form a separate phylogenetic group.

Three strains of Limnothrix (Cyanobacteria) isolated from Lake Kastoria, Greece, were characterized based on their morphological features and 16S rRNA gene sequences. The Limnothrix isolates 007a, 165a, and 165c can morphologically be assigned to Limnothrix redekei (Van Goor) Meffert. The 16S rRNA gene of the Limnothrix strains showed a 99% similarity to the 16S rRNA gene of Planktothrix sp. FP1. Limnothrix redekei strains 165a, 165c, 007a and Planktothrix sp. FP1 formed a separate cluster in the cyanobacterial 16S rRNA gene tree. It was distinct from the Pseudanabaena cluster, which included the other Limnothrix strains isolated from northern temperate lakes. This is the first report on the phylogeny of L. redekei strains originating from a Mediterranean lake (southern Europe) and provides new data about the genus Limnothrix.

Toxin extraction from an Anabaenopsis milleri--dominated bloom.

This paper reports the presence of microcystin type toxins in extracts of a natural bloom of cyanobacteria composed predominantly of Anabaenopsis milleri Woronichin. The toxins have been extracted, purified and compared to microcystin-LR. The LD50 of A. milleri bloom material was 600-1500 mg lyophilized cells/kg body weight. Symptoms and pathological signs of poisoning in mice were characteristic of cyanobacterial hepatotoxins, with enlarged darkened livers with weights of 8-10% of the total body weight. Thin-layer chromatography of the extract resulted in one toxic band, which was separated from pigments and 280-nm absorbing compounds. The toxic fraction was further separated using reversed phase high performance liquid chromatography and one toxic band was recovered. This fraction yielded a single, toxic peak with a retention time of 11.3 min after high performance liquid chromatography. The absorption spectrum of the purified toxin had a maximum at 238-240 nm and was characteristic of cyanobacterial hepatotoxic peptides. Comparison of the chromatographic behaviour of the purified toxin with microcystin-LR on reversed phase and on internal surface reversed phase, high performance liquid chromatography indicated that an A. milleri bloom material toxin was a microcystin type toxin and it is highly likely that the purified toxin is microcystin-LR.

Computer modelling of the 3-dimensional structures of the cyanobacterial hepatotoxins microcystin-LR and nodularin.

The 3-dimensional structures of two cyanobacterial hepatotoxins microcystin-LR, a cyclic heptapeptide and nodularin, a cyclic pentapeptide, and the novel amino acid ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid) were constructed, and optimized using the CHEM-X molecular mechanics program. The peptide rings were planar and of rectangular shape. Optimized ADDA formed a U-shape and a difference in the orientation of ADDA with respect to the peptide ring of the two hepatotoxins was observed.

Variations in ribulose 1,5-bisphosphate carboxylase protein levels, activities and subcellular distribution during photoautotrophic batch culture of Chlorogloeopsis fritschii.

Ribulose 1,5-bisphosphate (RuBP) carboxylase is present in the cytoplasm and carboxysomes (polyhedral bodies) of the cyanobacterium Chlorogloeopsis fritschii. In vitro enzyme activities have been measured throughout photoautotrophic batch culture, together with RuBP carboxylase protein concentrations, determined by rocket immunoelectrophoresis. Enzyme activities and protein levels in the cytoplasmic and carboxysomal fractions varied in an apparently inverse manner during growth. The RuBP carboxylase activities per unit enzyme protein were maximal in late lag phase/early exponential phase for both cellular enzyme pools. Both rates per unit enzyme protein declined during exponential phase, cytoplasmic enzyme activity remaining consistently higher than that of the carboxysomal enzyme. Activities per unit cytoplasmic and carboxysomal enzyme protein showed very low, similar rates in late stationary phase and death phase. Dialysis experiments indicated that such changes were not due to interference in activity assays by soluble endogenous effectors. Major shifts in the subcellular distribution of RuBP carboxylase protein were found versus culture age, enzyme protein levels being predominantly carboxysomal in lag phase, mainly soluble in exponential phase and then mainly carboxysomal again in stationary/death phase. The data are discussed in terms of carboxysome function and the question of control of RuBP carboxylase synthesis in cyanobacteria.

Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii.

Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.