Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the Bacillus subtilis genome project, we found that a mutant with inactivation of iolT (ydjK) exhibited a growth defect on myo-inositol as the sole carbon source. The putative product of iolT exhibits significant similarity with many bacterial sugar transporters in the databases. In B. subtilis, the iolABCDEFGHIJ and iolRS operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol. Among the iol genes, iolF was predicted to encode an inositol transporter. Inactivation of iolF alone did not cause such an obvious growth defect on inositol as the iolT inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single iolT inactivation. Determination of inositol uptake by the mutants revealed that iolT inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable. These results, as well as the K m and V max values for the IolT and IolF inositol transporters, indicated that iolT and iolF encode major and minor inositol transporters, respectively. Northern and primer extension analyses of iolT transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by A RNA polymerase and negatively regulated by IolR as well. The interaction between IolR and the iolT promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the iol divergon.
Accumulating evidence suggests that the delivery of human adipose tissue-derived stromal cells (hASCs) has great potential as regenerative therapy. This was performed to develop a method for expanding hASCs by reducing the amount of serum required. We demonstrate that hASCs were able to expand efficiently in media containing 2% serum and fibroblast growth factor-2. These cells, or low serum cultured hASCs (hLASCs), expressed cell surface markers similar to those on bone marrow-derived mesenchymal stem cells, and could be differentiated into cells of mesenchymal lineage. Of interest, hLASCs secreted higher levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) than hASCs cultured in 20% serum (hHASCs). Moreover, hLASC-conditioned media significantly increased endothelial cell (EC) proliferation and decreased EC apoptosis compared to that obtained from hHASCs or control media only. Antibodies against VEGF and HGF virtually negated these effects. When hASCs were administered into the ischemic hindlimbs of nude rats, hLASCs improved blood flow, increased capillary density, and raised the levels of VEGF and HGF in the muscles as compared with hHASCs. In conclusion, we demonstrate a novel low serum culture system for hASCs, which may have great potential in regenerative cell therapy for damaged organs in the clinical setting.
The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.
Many membrane proteins are proposed to work as oligomers; however, the conclusion is sometimes controversial, as for β2-adorenergic receptor (β2AR), which is one of the best-studied family A G-protein-coupled receptors. This is due to the lack of methods for easy and precise detection of the oligomeric state of membrane proteins on living cells. Here, we show that a combination of the coiled-coil tag-probe labeling method and spectral imaging enable a stoichiometric analysis of the oligomeric state of membrane proteins on living cells using monomeric, dimeric, and tetrameric standard membrane proteins. Using this method, we found that β2ARs do not form constitutive homooligomers, while they exhibit their functions such as the cyclic adenosine 5'-monophosphate (cAMP) signaling and internalization upon agonist stimulation.
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