Expression of Trisk 51, agrin and nicotinic-acetycholine receptor ε-subunit during muscle development in a novel three-dimensional muscle-neuronal co-culture system

The purpose of our study was to create functional muscle tissue in vitro and to investigate the influence of organotypic neuronal slice cultures from rat spinal cord on the differentiation and function of primary rat myoblasts in a novel three-dimensional culture system. Three-dimensional muscle-neuronal cultures were established by co-cultivating primary rat skeletal muscle cells of newborn rats with organotypic slice cultures of the spinal cord prepared from isogenic rats in a fibrin matrix. These constructs were cultured for up to 4 weeks. Differentiation and fusion of the myoblasts to myofibers was evaluated by analyzing the expression pattern and localization of muscle- and neuron-specific markers. The fibrin matrix provided a suitable environment for three-dimensional myoblast culture. Co-culturing of organotypic spinal cord slices with myoblasts induced the formation of spontaneously contracting multinuclear and parallel-aligned myofibers. Pharmacological tests suggested the formation of neuromuscular junctions. The analysis of neural agrin expression and myogenic desmin, myogenin, MyoD, Trisk 51, and nicotinic-acetycholine receptor (nACh-receptor) -subunit expression revealed the differentiation of the myoblasts to myofibers. The presented novel three-dimensional co-culture system allows the in vitro investigation of myoblast differentiation and neuron-myoblast interaction. Our results suggest the existence of an alternative pathway for the maturation of the nAChR -subunit to the -subunit without neural agrin activity.A.D.B. and J.P.B. contributed equally to this studyThis work was supported by a major grant from the State of Baden-Württemberg within the scope of the Valley TEC (Valley Tissue Engineering Center)     

Human herpesvirus-8-encoded signalling ligands and receptors

Analysis of the genome of human herpesvirus 8 (HHV-8) led to the discovery of several novel genes, unique among the characterized gammaherpesviruses. These include cytokines (interleukin-6 and chemokine homologues), two putative signal-transducing transmembrane proteins encoded by genes K1 and K15 at the genome termini, and an OX-2 (CD200) receptor homologue that had not previously been identified in a gammaherpesvirus. HHV-8 also specifies a diverged version of the gammaherpesvirus-conserved G protein-coupled chemokine receptor (vGCR) and a latently expressed protein unique to HHV-8 specified by open reading frame (ORF) K12. These cytokine and receptor homologues mediate signal transduction or modulate the activities of other endogenous cytokines and receptors to enhance viral productive replication, regulate latent-lytic switching, evade host attack, or mediate cell survival. The viral signalling ligands and receptors are also potential contributors to virus-associated diseases, Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, and so represent potentially important targets for therapeutic and antiviral drugs. Understanding these proteins' modes of action and functions in viral biology and disease is therefore of considerable importance, and the subject of this review.     

cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai

An alginate lyase, termed HdAly in the present paper, was isolated from the hepatopancreas of abalone, Haliotis discus hannai, by ammonium sulfate fractionation, followed by TOYOPEARL CM-650M column chromatography. Enzymatic properties of HdAly were similar to those of previously reported Haliotis and Turbo poly(M) lyases, e.g., it preferentially degraded a poly(beta-D-mannuronate)-rich substrate with an optimal pH and temperature at pH 8.0 and 45 degrees C, respectively. In order to determine the primary structure of abalone lyase that is still poorly understood, cDNAs for HdAly were cloned by PCR from the abalone hepatopancreas cDNA library and sequenced. From the nucleotide sequences of the cDNAs, the sequence of 909 bp in total was determined, and the amino acid sequence of 273 residues was deduced from the translational region of 822 bp locating at nucleotide positions 27-848. The N-terminal region of 16 residues, except for the initiation Met in the deduced sequence, was regarded as the signal peptide since it was absent in the HdAly protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretary proteins. This suggests that HdAly is initially produced as a precursor possessing the signal peptide in hepatopancreatic cells and then secreted into digestive tract as the mature form. Thus, the mature HdAly was regarded to consist of 256 residues with the calculated molecular mass of 28895.5 Da. The amino acid sequence of HdAly showed 85 and 28% identity to those of Turbo cornutus alginate lyase SP2 and the C-terminal region of Chlorella virus lyase-like protein CL2, respectively, while it showed no significant identity to those of any bacterial alginate lyases. In order to provide the basis for the structure-function studies and various applications of the abalone lyase, a bacterial expression system was constructed by means of the HdAly-cDNA and pET-3a expression plasmid. Although the active recombinant HdAly was hardly produced at a cultivation temperature 37 degrees C in Escherichia coli BL21 (DE3), a small amount of soluble and active enzyme could be produced when the temperature was lowered to 19 degrees C.     

A role for programmed cell death during early neurogenesis in xenopus

In vertebrates, little is known on the role of programmed cell death (PCD) occurring within the population of dividing neural precursors and newly formed neuroblasts during early neural development. During primary neurogenesis, PCD takes place within the neuroectoderm of Xenopus embryos in a reproducible stereotypic pattern, suggesting a role for PCD during the early development of the CNS. We find that the spatio-temporal pattern of PCD is unaffected in embryos in which cell proliferation has been blocked and whose neuroecotoderm contains half the normal number of cells. This shows that PCD is not dependent on cell division. It further suggests that PCD does not solely function to regulate absolute cell numbers within the neuroectoderm. We demonstrate that PCD can be reproducibly inhibited in vivo during primary neurogenesis by the overexpression of human Bcl-2. Following PCD inhibition, normal neurogenesis is disrupted, as seen by the expansion of the expression domains of XSox-2, XZicr-2, XNgnr-1, XMyT-1, and N-Tubulin, XNgnr-1 being the most affected. PCD inhibition, however, did not affect the outcome of lateral inhibition. We propose, then, that PCD regulates primary neurogenesis at the level of neuronal determination.     

Molecular ordering of cellulose after extraction of polysaccharides from primary cell walls of Arabidopsis thaliana: a solid-state CP/MAS (13)C NMR study

Solid-state CP/MAS 13C NMR spectroscopy was used to determine the effects of three different sequential extraction procedures, used to remove non-cellulosic polysaccharides, on the molecular ordering of cellulose in a cell-wall preparation containing mostly primary cell walls obtained from the leaves of the model dicotyledon, Arabidopsis thaliana. The extractions were 50 mM trans-1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid (CDTA) and 50 mM sodium carbonate (giving Residue 1); 50 mM CDTA, 50 mM sodium carbonate and 1 M KOH (giving Residue 2); and 50 mM CDTA, 50 mM sodium carbonate and 4 M KOH (giving Residue 3). The molecular ordering of cellulose in Residue 1 was similar to that in unextracted walls: the cellulose was almost all crystalline, with 43% of molecules contained in crystallite interiors and similar proportions of the triclinic (I(alpha)) and monoclinic (I(beta)) crystal forms. Residue 2 was partly decrystallized and the remaining crystallites were mostly in the I(beta) form. Residue 3 was a mixture of cellulose II, cellulose I and amorphous cellulose. The presence of signals at 100.0 and 102.3 ppm in the spectra of Residues 1 and 2, but not of unextracted cell walls, suggested that the extractions giving these residues caused some of the non-cellulosic polysaccharides, possibly xyloglucans and galactoglucomannans, to become relatively well ordered, for example through interactions with cellulose crystallite surfaces.     

Structural and immunological aspects of Polybia scutellaris Antigen 5

Vespid venoms contain Antigen 5, an important allergen whose primary structure and immunological behavior have been extensively studied from venoms of vespids of the Northern Hemisphere. We report herein structural and immunological aspects of Antigen 5 from Polybia scutellaris subspecies rioplatensis (vulgar name: camoati) found in South America. Mast cell degranulation, histamine release, and IgE induction experiments performed in mice allow us to suggest that P. scutellaris Antigen 5 is a variant with reduced IgE response and anaphylactic activity. Sequence data indicate that the protein has a 72.5-90.3% similarity to that of members of the vespid Antigen 5 family with an already known primary structure. Moreover, results suggest that the protein-a new member of an extracellular protein superfamily-could be a good candidate for immunotherapy related to vespid allergy.     

Formulation of a coupled mechanism between solute diffusion,phosphatase-kinase reactions and membrane potentials for the primary active transport of phosphorylated substrates through biological membranes

Coupled interrelations occurring between a phosphatase/kinase reaction sequence acting in unstirred layers and on both sides of a charged biomembrane pore structure are presented as a plausible kinetic model for the primary active transport of phosphorylated molecules. Simulations conducted at the cell level and with credible numerical values demonstrate that the enzymes positions strongly regulate the membrane permeability for the transported substrate. Depending on both the enzymes positions (more or less far from the membrane) and the membrane charges, the membrane may appear either impervious, either permeable or able to actively transport a phosphorylated substrate. Globally all happens as if, in function of the enzymes positions, a permanent pore may be regulated, changing from a more closed to a more open conformation.     

Comparative Molecular Evolution of Primary (<Emphasis Type="BoldItalic">Buchnera</Emphasis>) and Secondary Symbionts of Aphids Based on Two Protein-Coding Genes

A+T content, phylogenetic relationships, codon usage, evolutionary rates, and ratio of synonymous versus non-synonymous substitutions have been studied in partial sequences of the atpD and aroQ/pheA genes of primary (Buchnera) and secondary symbionts of aphids and a set of selected non-symbiotic bacteria, belonging to the five subdivisions of the Proteobacteria. Compared to the homologous genes of the last group, both genes belonging to Buchnera behave in a similar way, showing a higher A+T content, forming a monophyletic group, a loss in codon bias, especially in third base position, an evolutionary acceleration and an increase in the number of non-synonymous substitutions, confirming previous results reported elsewhere for other genes. When available, these properties have been partly observed with the secondary symbionts, but with values that are intermediate between Buchnera and free living Proteobacteria. They show high A+T content, but not as high as Buchnera, a non-solved phylogenetic position between Buchnera, and the other γ-Proteobacteria, a loss in codon bias, again not as high as in Buchnera and a significant evolutionary acceleration in the case of the three atpD genes, but not when considering aroQ/pheA genes. These results give support to the hypothesis that they are symbionts at different stages of the symbiotic accommodation to the host.     

Vegetation development on pumice at Mount St. Helens, USA

This study explores early vegetation development on pumice at Mount St.Helens. We monitored species annually in a grid of 200 contiguous100-m² plots between 1989 and 1999. Of interestwere how vegetation changed and if it became more homogeneous over time.Speciesrichness and cover increased annually. Diversity(H) stabilized by 1996 and began to decline aslong-livedstress-tolerant species such as Agrostis pallens, Carexmertensii and Penstemon cardwellii began todominate. Protected sites had more species and higher cover than did exposedones. Plots next to relict vegetation had more species and cover than diddistant plots. The vegetation initially was dominated by species with gooddispersal, but subsequently those with poor dispersal became dominant. Wecompared species expansion patterns to a model based on random colonization.Theresults implied that populations with poor dispersal derived from a fewcolonists that then produced seeds for local expansion. Detrendedcorrespondenceanalysis showed a pronounced shift in species composition. This analysis alsoshowed that species composition was becoming more homogeneous over time.However, significant heterogeneity remained and some plots are diverging fromothers. As yet, this vegetation is not developing towards a regional vegetationtype. Rather, an unusual community with Agrostis spp.,Carex spp., Penstemon cardwellii, Lupinuslepidus, Anaphalis margaritacea and Salixcommutata has developed. The accumulation phase of primarysuccessionis nearly complete. The next phase, in which vertical structure develops asSalix and conifers mature, has scarcely begun. It shouldbemarked by the invasion of forest understory species and loss of subalpinemeadowspecies. Assembly rules based on biotic interactions may then become evident.