Adult mesenchymal stem cells: potential for muscle and tendon regeneration and use in gene therapy

The expansion potential and plasticity of stem cells, adult or embryonic, offer great promise for their use in medical therapies. Recent provocative data suggest that the differentiation potential of adult stem cells may extend to lineages beyond those usually associated with the germ layer of origin. In this review, we describe recent developments related to adult stem cell research and in particular, in the arena of mesenchymal stem cell (MSC) research. Research demonstrates that transduced MSCs injected into skeletal muscle can persist and express secreted gene products. The ability of the MSC to differentiate into cardiomyocytes has been reported and their ability to engraft and modify the pathology in infarcted animal models is of great interest. Research using MSCs in tendon repair provides information on the effects of physical forces on phenotype and gene expression. In turn, MSCs produce changes in their matrix environment in response to those biomechanical forces. Recent data support the potential of MSCs to repair tendon, ligament, meniscus and other connective tissues. Therapeutic applications of adult stem cells are approaching clinical use in several fields, furthering the possibility to regenerate damaged and diseased tissue.     

SED1 gene length and sequence polymorphisms in feral strains of Saccharomyces cerevisiae

The SED1 gene (YDR077W), coding for the major cell wall glycoprotein of Saccharomyces cerevisiae stationary-phase cells, contains two blocks of tandem repeat units located within two distinct regions of the nucleotide sequence. A PCR survey of the SED1 open reading frames (ORFs) of 186 previously uncharacterized grape must isolates of S. cerevisiae yielded 13 PCR profiles arising from different combinations of seven SED1 length variants in individuals homozygous or heterozygous for the gene. Comparison of the nucleotide sequences of a group of representatives of each of the seven length variants with those of S288C and the type strain, CBS1171, unequivocally identified them as SED1 alleles and provided evidence for the presence of two minisatellite-like sequences, variable in length, within the ORF of an S. cerevisiae gene. The segregation analyses of the SED1 length variants and other genetic markers in 13 isolates representative of each PCR profile suggested that molecular mechanisms involved in minisatellite expansion and contraction may be responsible for SED1 heterozygosities within a population of homothallic must isolates of S. cerevisiae.     

不同季节和冬眠时相中达乌尔黄鼠视前区温敏神经元特性和下丘脑内NA代谢的变化

比较了不同季节和冬眠时相中达乌尔黄鼠 (Citelleusdauricus)下丘脑内去甲肾上腺素 (noradrenaline ,NA)代谢和视前区 (POA)脑片中各类温敏神经元的比例、温度敏感性、放电活动的临界温度及下限温度 .结果表明 :与夏季动物相比 ,( 1)冬眠各时相中POA温敏神经元的比例和温敏性产生了与冬眠体温调节特性相关的适应性改变 ;( 2 )冬季和冬眠中POA神经元放电的下限温度和温敏神经元活动的临界温度均显著下移 ;( 3 )冬眠中POA神经元对NA反应的敏感性增高 ,冷敏神经元对NA的反应从夏季的抑制型转变为冬眠时的兴奋型 ;( 4)入眠和深冬眠时下丘脑内NA的含量和代谢水平下降 ,出眠时代谢水平升高 .这些变化可能解释动物入眠时主动降低体温和出眠时从深低体温中快速地升温的温度调节机理 .     

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.      

NMR conformational studies on a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor

The combined use of several nuclear magnetic resonance and restrained molecular dynamics techniques allowed the formulation of a molecular model for the preferred solution conformation of a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor. In the model, the conformation of the 20-membered tocin ring, with the two Cys¹ and Cys⁶ residues bridged by a disulphide bond, is very close to that observed for isolated ocytocin in the solid state; in addition, a type II β-turn is postulated for the 7-10 segment of the acyclic tail containing the Lys¹¹-Arg¹² processing site, and connecting ocytocin to the neurophysin domain, while the C-terminal 13-20 segment of the molecule is believed to assume a helical structure. This particular structural organization could be important in participating as the favorable conformation for optimal substrate-enzyme active site recognition and processing by specific endoproteases. © 1996 John Wiley & Sons, Inc.     

Characterization of Channel-Forming Activity in Muscle Biopsy from a Porin-Deficient Human Patient

A bioptic specimen from the muscles of a patient suffering from severe myopathy was inspected for the presence of human porin 31HL. Western blotting suggested that the specimen was free of the most abundant eukaryotic porin 31HL (HVDAC1). The specimen was treated with detergent and the soluble protein fraction was passed through a dry hydroxyapatite column. The passthrough of this column was inspected for channel formation in artificial lipid-bilayer membranes. The channel observed under these conditions had a single-channel conductance of about 2.5 nS in 1 M KCl, was cation selective, and was found to be virtually voltage independent. Experiments with a control specimen from a healthy human being, without any indication for muscle myopathy, revealed the presence of the voltage-dependent porin 31HL in the sample. It is discussed whether the patient's bioptic specimen contained another human porin, which has not been studied to date in its natural environment.     

Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3.

Previous studies using selectively modified pro-ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro-ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a beta-turn structure. We have used various peptide analogues of the pro-ocytocin-neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N-Met) modification, a secondary structure beta-turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side-chains were oriented in opposite directions; (c) the amino acid present at the P'1 position is important in the enzyme-substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro-ocytocin/neurophysin large substrate over that of the smaller one pGlu-Arg-Thr-Lys-Arg-methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro-ocytocin-related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.