PubMed · 15190175
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Esther Landhuis. 2004-06-09. Counterattack.. https://doi.org/10.1126/sageke.2004.23.nf57
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Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.
Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.
Vitellogenins (Vtgs) are yolk precursor proteins in oviparous species and are cleaved into three portions-lipovitellin I (LVI), phosvitin (PV), and lipovitellin II (LVII)-in oocytes in vertebrates. In the present study, we found that the zebrafish genome contains at least seven vtg genes (vtg1-7) encoding heterogeneous vitellogenins with three distinct types of Vtgs: type I (Vtg1, 4-7) contains all the three major portions but lacks the C-terminal half of LVII; type II (Vtg2) is the only one including intact three portions; type III (Vtg3) lacks both PV and the LVII C-terminal half. The seven vtgs were located in two different chromosomes: one (vtg3) in LG11 and the rest closely linked in LG22, probably arisen from local gene duplication events. All of the seven vtgs are predominantly expressed in female liver and can be induced in male liver by 17beta-estradiol (E2). The level of vtg1 mRNA was about 100x and 1000x higher than those of vtg2 and vtg3 mRNAs. We also found vtg mRNAs in several non-liver tissues, but the expression level is generally <10% of that in the liver. In situ hybridization experiments confirmed that the extrahepatic expression was actually in adipocytes associated with several organs such as the intestine, ovary, and E2-induced testis.