Myosatellite cells of Cyprinus carpio (Teleostei) in vitro: isolation,recognition and differentiation

Summary We have developed a method for the dissociation and purification of myosatellite cells from white epaxial muscle of carp. The dissociated myosatellite cells were identified by their morphology, their ultrastructure, the formation of multinucleated myotubes containing myofibrils and the immunocytochemical demonstration of desmin. Desmin and 5-bromo-2-deoxyuridine (BrdU) were used to identify terminally differentiated and proliferating myosatellite cells, respectively. The in vitro behavior of myosatellite cells dissociated from carp of 5 cm standard length differed from that described for myosatellite cells of mammals and birds. No substantial proliferation of the myosatellite cells could be observed. Most cells were differentiated (desmin-positive, BrdU-negative) 17 h after plating, regardless of the medium used. This indicates that the investigated white epaxial muscle of carp of 5 cm standard length contains subpopulations of myosatellite cells, arrested at various stages of differentiation.     

Purification and some properties of the methyl-CoM reductase of Methanothrix soehngenii

Abstract The methyl-CoM reductase from Methanothrix soehngenii was purified 18-fold to apparent homogeneity with 50% recovery in three steps. The native molecular mass of the enzyme estimated by gel-fitration was 280 kDa. SDS-polyacrylamide gel electrophoresis revealed three protein bands corresponding to M _r 63 900, 41 700 and 30 400 Da. The methyl-coenzyme M reductase constitutes up to 10% of the soluble cell protein. The enzyme has K _m apparent values of 23 μM and 2 mM for N -7-mercaptoheptanoylthreonine phosphate (HS- HTP = component B ) and methyl-coenzyme M (CH₃CoM) respectively. At the optimum pH of 7.0 60 nmol of methane were formed per min per mg protein.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.     

Callus culture ofPterocladia capillacea (Rhodophyta) and analysis of cell wall polysaccharides

Calluses induced fromPterocladia capillacea have been kept in culture for more than three years. They exhibit a fast growth rate, owing to the release of single cells, which in turn develop into new callus. The effect of various media and culture conditions upon growth was investigated. In order to confirm the identity of the callus cells, a 0,45 mg incoculum was grown that yielded 15 g dried callus within six weeks. Polysaccharides from this material (5.5 g) were analysed by¹³C NMR spectroscopy. This produced a spectrum typical of agar and very similar to the one obtained for agar extracted fromP. capillacea plants. However, the callus agar displayed no gel-forming properties, even after alkali modification.author for correspondence     

C1q,a collagen-like complement subcomponent,in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase)

C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

中国3个地区汉族人群补体C6的遗传多态性研究

运用聚丙烯酰胺凝胶等电聚焦(PAGIF)和免疫吸引技术,研究了3个地区汉族人群的C6多态性。得到的基因频率如下:漳州市——C6*A:0.4634、C6*B:0.5000、C6*R:0.0366(C6*B2:0.0317);成都市——C6*A:0.4975,C6*B:0.4484,C6*R:0.0545(C6*B2:0.0395);哈尔滨市——C6*A:0.4708,C6*B:0.5219,C6*R:0.0073(C6*B2:0.0073)。蒙古人种的C6*A频率一般都低于0.5,高加索人种的C6*A频率一般都高于0.6。黑人则介于两者之间。蒙古人种与高加索人种的另一个区别在于前者的C6*B2频率在0.03到0.07之间,而后者几乎没有C6*B2。     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.