Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture

Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage.     

Ganglioside GD3 Enhances Adhesion Signals and Augments Malignant Properties of Melanoma Cells by Recruiting Integrins to Glycolipid-enriched Microdomains

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130^Cas, and paxillin when treated with fetal calf serum than GD3− cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin β1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin β1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin β1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.     

The short apical membrane half-life of rescued {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {Delta}F508-CFTR in polarized human airway epithelial cells

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the apical plasma membrane. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the DeltaF508 mutation reduces the half-life of DeltaF508-CFTR in the apical plasma membrane. Because DeltaF508-CFTR retains some Cl(-) channel activity, increased expression of DeltaF508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued DeltaF508-CFTR that lead to the decreased apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the DeltaF508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.     

Histochemical studies on the morphology of the Golgi apparatus in the neurons of supraoptic and paraventricular nuclei of the normal and dehydrated rabbit (application of the thiamine pyrophosphatase method)

Summary Detailed histochemical studies have been conducted on the morphology of the Golgi apparatus by applying the thiamine pyrophosphatase technique (Novikoff and Goldfisher, 1961) to the neurons of supraoptic and paraventricular nuclei of normal and dehydrated rabbits. The neurons in both nuclei were classified into five categories on the basis of the morphology of the Golgi apparatus. The number of cells in individual categories were counted to evaluate the percentage of each category in the whole nucleus.Neurons have many vesicles which show the tendency to form clusters. Such clusters are present also in the basal bodies. The Golgi apparatus is localized near one side of the nucleus in many neurons. The neurons indicate phasic activity of resting, anabolic and catabolic stages under normal conditions.During dehydration, the Golgi apparatus went through the three stages of network formation, the increase of the budding-off process and later on disintegration. The supraoptic nucleus reacted to the TPPase test more severely than the paraventricular nucleus, whereas the former went through the stages more slowly than the latter. The paraventricular nucleus also revealed sensitivity to osmotic stress.     

Development and characterisation of microsatellite loci in Farfugium japonicum (Asteraceae)

Eight microsatellite loci for the perennial herb Farfugium japonicum, including the rheophytic variety luchuense endemic to riparian areas of the Ryukyu Islands, Japan, were isolated and characterised. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.344–0.885 and 0.121–0.754, respectively, from 69 individuals in one population. Six loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). The primers amplifying microsatellite sequences in F. japonicum may provide a population genetics tool useful in the establishment of a conservation strategy.     

Microsatellite polymorphism in the human heme oxygenase-1 gene promoter and its application in association studies with Alzheimer and Parkinson disease

Oxidative stress has been suggested to be involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer disease (AD) and Parkinson disease (PD). Heme oxygenase-1 (HO-1), a key enzyme in heme catabolism, also functions as an antioxidant enzyme. Here, we show that a (GT)_n repeat in the human HO-1 gene promoter region is highly polymorphic, although no particular alleles are associated with AD or PD. This newly identified genetic marker should allow us to study the possible involvement of HO-1 in certain human diseases. Received: 5 November 1996 / Accepted: 18 February 1997     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.