Plasminogen activator inhibitor-1 deficiency suppresses osteoblastic differentiation of mesenchymal stem cells in mice

Plasminogen activator inhibitor-1 (PAI-1) is known as an inhibitor of fibrinolytic system. Previous studies suggest that PAI-1 is involved in the pathogenesis of osteoporosis induced by ovariectomy, diabetes, and glucocorticoid excess in mice. However, the roles of PAI-1 in early-stage osteogenic differentiation have remained unknown. In the current study, we investigated the roles of PAI-1 in osteoblastic differentiation of mesenchymal stem cells (MSCs) using wild-type (WT) and PAI-1-deficient (PAI-1 KO) mice. PAI-1 mRNA levels were increased with time during osteoblastic differentiation of MSCs or mesenchymal ST-2 cells. However, the increased PAI-1 levels declined at the mineralization phase in the experiment using MC3T3-E1 cells. PAI-1 deficiency significantly blunted the expression of osteogenic gene, such as osterix and alkaline phosphatase enhanced by bone morphogenetic protein (BMP)-2 in bone marrow-derived MSCs (BM-MSCs), adipose-tissue-derived MSCs (AD-MSCs), and bone marrow stromal cells of mice. Moreover, a reduction in endogenous PAI-1 levels by small interfering RNA significantly suppressed the expression of osteogenic gene in ST-2 cells. Plasmin did not affect osteoblastic differentiation of AD-MSCs induced by BMP-2 with or without PAI-1 deficiency. PAI-1 deficiency and a reduction in endogenous PAI-1 levels did not affect the phosphorylations of receptor-specific Smads by BMP-2 and transforming growth factor-β in AD-MSCs and ST-2 cells, respectively. In conclusion, we first showed that PAI-1 is crucial for the differentiation of MSCs into osteoblasts in mice.     

Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.     

Levels of endogenous ethylene, carbon dioxide, and soluble pectin, and activities of pectin methylesterase and polygalacturonase in ripening tomato fruits

In 4 cultivars of tomato (Lycopersicon esculentum Mill.), theearly detachment of fruits advanced ripening and considerablyreduced the threshold value of endogenous C₂H₄. This indicatesa supply from the vegetative parts of (a) labile ripening-inhibitingsubstance(s) antagonizing the action of C₂H₄. The endogenous level of CO₂ increased shortly after the risein C₂H₄, and maximum levels of C₂H₄ and CO₂ occurred almostsimultaneously. The activity of PE showed no connection with ripening, but PGactivity did not occur until the onset of ripening. However,this activity increased at considerably higher C₂H₄ concentrationsthan the rise in WSP, and was independent of the possible presenceof ripening inhibitor(s). Hence PG is considered not to be involvedin the primary events leading to fruit ripening. Exposure of fruits to different C₂H₄ concentrations in the ambientatmosphere also showed PG activity to increase only after therise in WSP had started. Other pectin degrading or synthesizingenzymes may be involved. In the non-ripening Rin mutant of cv. Rutgers, no rise occurredin C₂H₄, CO₂, WSP, and PG activity. ¹ Present address: Department of Agricultural Chemistry, Facultyof Agriculture, Kochi University, Otsu 200 Monobe, Nangoku City,Kochi Prefecture 783, Japan. (Received February 16, 1978; )     

Cytosolic r{beta}-Cyanoalanine Synthase Activity Attributed to Cysteine Synthases in Cocklebur Seeds. Purification and Characterization of Cytosolic Cysteine Synthases

The activity of rß-cyanoalanine synthase (CAS, EC4.4.1.9 [EC] ) in cotyledons of cocklebur seeds (Xanthium penn-sylvanicumWallr.) was detected both in the soluble and particulate fractions.The CAS activity of the soluble fraction (cytosolic CAS activity)was 10 times higher than that of the particulate fraction. TheCAS activity of the particulate fraction was confirmed to belocalized in the mitochondria. Both enzymatic activities wereclearly separated by non-denaturing PAGE. The enzyme with cytosolicCAS activity has been extensively purified and separated intothree different forms designated as cyt-1, cyt-2, and cyt-3.According to the SDS-PAGE analysis, the three enzymes are estimatedto be a homodimer composed of 35-kDa sub-units. The purifiedenzymes showed CS activity. Partial amino acid sequences ofcyt-1 were determined and had a high homology with cysteinesynthases (CS, EC 4.2.99.8 [EC] ) from other plant sources. The catalyticaction of the purified CSs in converting cyanide and cysteineinto H₂S and rß-cyanoalanine was confirmed by thedetection of significant ¹⁴CN incorporation into rß-cyanoalanine.These results indicated that cytosolic CAS activity is due tocytosolic CS and suggested that the CAS activity of CS is likelyto be involved in cyanide metabolism in plant tissues. (Received January 7, 1998; Accepted March 16, 1998)     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain

The epidermal growth factor receptor plays crucial roles throughout the development of multicellular organisms, and inappropriate activation of the receptor is associated with neoplastic transformation of many cell types. The receptor is thought to be activated by ligand-induced homodimerisation. Here, however, we show by chemical cross-linking and sucrose density-gradient centrifugation that in the absence of bound ligand the receptor has an ability to form a dimer and exists as a preformed dimer on the cell surface. We also analysed the receptor dimerisation by inserting cysteine residues at strategic positions about the putative alpha-helix axis of the extracellular juxtamembrane region. The mutant receptors spontaneously formed disulphide bridges and transformed NIH3T3 cells in the absence of ligand, depending upon the positions of the cysteine residue inserted. Kinetic analyses of the disulphide bonding indicate that EGF binding induces flexible rotation or twist of the juxtamembrane region of the receptor in the plane parallel with the lipid bilayer. The binding of an ATP competitor to the intracellular domain also induced similar flexible rotation of the juxtamembrane region. All the disulphide-bonded dimers had flexible ligand-binding domains with the same biphasic affinities for EGF as the wild-type. These results demonstrate that ligand binding to the flexible extracellular domains of the receptor dimer induce rotation or twist of the juxtamembrane regions, hence the transmembrane domains, and dissociate the dimeric, inactive form of the intracellular domains. The flexible rotation of the intracellular domains may be necessary for the intrinsic catalytic kinase to become accessible to the multiple tyrosine residues present in the regulatory domain and various substrates, and may be a common property of many cell-surface receptors, such as the insulin receptor.     

Fatty acids increase the circulating levels of oxidative stress factors in mice with diet-induced obesity via redox changes of albumin

Plasma concentrations of free fatty acids are increased in metabolic syndrome, and the increased fatty acids may cause cellular damage via the induction of oxidative stress. The present study was designed to determine whether the increase in fatty acids can modify the free sulfhydryl group in position 34 of albumin (Cys34) and enhance the redox-cycling activity of the copper-albumin complex in high-fat diet-induced obese mice. The mice were fed with commercial normal diet or high-fat diet and water ad libitum for 3 months. The high-fat diet-fed mice developed obesity, hyperlipemia, and hyperglycemia. The plasma fatty acid/albumin ratio also significantly increased in high-fat diet-fed mice. The increased fatty acid/albumin ratio was associated with conformational changes in albumin and the oxidation of sulfhydryl groups. Moreover, an ascorbic acid radical, an index of redox-cycling activity of the copper-albumin complex, was detected only in the plasma from obese mice, whereas the plasma concentrations of ascorbic acid were not altered. Plasma thiobarbituric acid reactive substances were significantly increased in the high-fat diet group. These results indicate that the increased plasma fatty acids in the high-fat diet group resulted in the activated redox cycling of the copper-albumin complex and excessive lipid peroxidation.     

Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum

The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.     

Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells

Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.