Differentiation of pericytes in culture is accompanied by changes in the extracellular matrix

Summary We have previously reported that pericytes derived from retinal and brain microvessels aggregate into nodules soon after reaching confluence. Nodule formation involves a reorganization of the cells resulting in the presence of sparse cells, confluent monolayers, multilayers, sprouts, and nodules within the same culture dish. Extracellular calcification occurs only within the nodules, demonstrating that pericytes are capable of undergoing osteogenic differentiation in culture and that this differentiation is related to nodule formation. Using immunofluorescence we have now studied the distribution of laminin, type IV collagen, type X collagen, and tenascin in pericyte cultures during nodule formation. These matrix macromolecules were also identified by a combination of biochemical techniques, including Northern blot hybridization, immunoblotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecule that seems to be related to type X collagen was demonstrated by the presence of a pepsin-resistant, collagenase-sensitive polypeptide of molecular weight approximately 45 kDa. The production of laminin, type X-related collagen, and tenascin by pericytes has not been previously reported. Our results suggest that the synthesis or distribution or both of these molecules is dependent on the state of pericyte differentiation. The expression of laminin, type IV collagen, and type X-related collagen was maximal in multilayer areas, sprouts, and nodules. Tenascin appeared homogeneously distributed in monolayer and multilayer areas; when calcified nodules were present, the anti-tenascin serum preferentially decorated a discrete area circumscribing the nodules. Tenascin and type X collagen have been found transiently in vivo preceding calcification; their possible role in this process is not known. Our results also suggest an association between laminin, type IV collagen, and calcification. The in vitro experimental system described here may help to clarify the role of matrix macromolecules in the calcification process.     

Glycerol and dihydroxyacetone metabolizing enzymes in fission yeasts of the genus Schizosaccharomyces

The only species of fission yeasts capable of growing on glycerol or dihydroxyacetone were Schizosaccharomyces pombe and S. malidevorans. When growing on glycerol or grown on glucose until it was exhausted, these species contained glycerol:NAD⁺ 2-oxidoreductase and dihydroxyacetone kinase but no glycerol kinase, consistent with utilization of glycerol via dihydroxyacetone. When grown to exhaustion of glucose, S. octosporus, S. slooffiae and S. japonicus contained dihydroxyacetone kinase but no glycerol:NAD⁺ 2-oxidoreductase or glycerol kinase. Prior to exhaustion of glucose in the medium, all species contained dihydroxyacetone kinase, all species except S. japonicus contained glycerol:NADP⁺ 2-oxidoreductase, and only S. pombe and S. malidevorans contained glycerol:NAD⁺ 2-oxidoreductase. Possible roles for the glycerol:NAD⁺ 2-oxidoreductase, glycerol:NADP⁺ 2-oxidoreductase and dihydroxyacetone kinase in metabolism of glycerol and dihydroxyacetone are discussed.Non-standard abbreviations DHA dihydroxyacetone - DHAK dihydroxyacetone kinase - DHAP dihydroxyacetone phosphate - GK glycerol kinase - G2DH-NAD glycerol - NAD⁺ 2-oxidoreductase - G2DH-NADP glycerol - NADP⁺ 2-oxidoreductase - MEA malt extract agar - YEP yeast extract phosphate medium     

Purification and properties of glycerol:NADP+ 2-oxidoreductase from Schizosaccharomyces pombe

Glycerol:NADP+ 2-oxidoreductase (EC 1.1.1.156) was isolated from Schizosaccharomyces pombe, purified and characterized. It had an Mr of 57,000, and SDS-PAGE revealed two polypeptides, of Mr 25,000 and 30,000. Its coenzyme requirement was satisfied exclusively by NADP. The pH optimum for glycerol oxidation was 9.5, for dihydroxyacetone reduction 6.0. Rates of oxidation with some structurally related diols were three- to six-fold lower than for glycerol, while glyceraldehyde and other carbonyl compounds showed negligible rates of reduction. Neither monovalent nor divalent cations activated the enzyme. Apparent Km and Vmax values were determined. The enzyme is similar to glycerol dehydrogenases isolated from Mucor javanicus and from Dunaliella parva but differs considerably from the glycerol:NAD+ 2-oxidoreductase of S. pombe.     

Flavones from Struthiola argentea with anthelmintic activity in vitro

Parasitic diseases caused by helminthes lead to significant health hazards to animals resulting in enormous economic impact. While a number of anthelmintics are currently available, all are encountering resistance and ones with a mode of action are needed. We report herein bioassay-guided isolation of three anthelmintic flavones 1-3, including the flavone, 5,6,2',5',6'-pentamethoxy-3',4'-methylenedioxyflavone (3) from the methanol extract of Struthiola argentea (Thymelaeaceae). The structure of 3 was elucidated by analysis of its 1D and 2D NMR and MS data. The two major flavones produced by this plant were also isolated and identified as yuankanin (4) and amentoflavone (5). A number of flavones related to the compounds isolated from S. argentea were acquired and tested to ascertain structure activity relationships. The isolation, structure, anthelmintic activity and structure activity relationships of the flavones are described. Compound 3 exhibited the most potent in vitro activity with 90% inhibition of larval motility at 3.1 microg/mL and compound 15 showed modest in vivo activity.     

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial‐targeted proteins (N‐mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N‐mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear‐ and mitochondrial‐encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N‐mt gene evolution in species with fast‐evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N‐mt substitutions at positions that directly contact mutated residues in mitochondrial‐encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N‐mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories.     

ConA的抗着床效应

本文用凝集素为探针,探索糖复合物在胚泡着床中的作用,报道了与甘露糖苷有专一结合的伴刀豆凝集素(ConA)有明显的抗小鼠胚泡着床作用。妊娠4d的小鼠,每只子宫角中注入Con A 25μg,22只子宫角中只有4只子宫角有胚泡着床,着床率为18.2％,与生理盐水对照组的着床率87.5％相比有明显差异。将相同剂量的Con A先与0.4mol/L α-甲基-D-甘露糖苷温育1—2h后再注入子宫,20只子宫角中有15只子宫角有胚泡着床,着床率提高到75％。用辣根过氧化物酶直接标记法证明,着床前子宫内膜细胞表面有Con A受体存在,并随着妊娠天数而增加,尤其是间质细胞,发情期时时为阴性反应,到着床期蜕膜细胞膜表面呈现出大量Con A受体。提示精复合物在着床中的重要作用。与甘露糖苷同样专一结合的,但寡糖结构专一性与Con A不同的豌豆凝集素注入子宫则无抗着床效应,着床率为85.7％。由此可以推测,N-连接的包含二个未被取代的或只在C-2位被取代的α-甘露糖苷的寡糖参于胚泡与子宫内膜相互作用的着床过程。     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.