We report the cloning and primary structure of the Drosophila insulin receptor gene (inr), functional expression of the predicted polypeptide, and the isolation of mutations in the inr locus. Our data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The INR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin‐binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The INR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension which contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C‐terminal domain undergoes cell‐specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C‐terminus) from the inr gene. These subunits assemble into mature INR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI‐3 kinase. It is likely that the two different isoforms of INR have different signaling potentials. Finally, we show that loss of function mutations in the inr gene, induced by either a P‐element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of inr appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are affected by inr mutations.
Aging in vivo and cell division in vitro are associated with telomere shortening. Several lines of evidence suggest that telomere length may be a good predictor of the long term replicative capacity of cells. To investigate the natural fate of chromosome telomeres of hematopoietic stem cells in vivo, we measured the telomere length of peripheral blood granulocytes from 11 fully engrafted bone marrow transplant recipients and from their respective donors. In 10 of 11 donor-recipient pairs, the telomere length was significantly reduced in the recipient and the extent of reduction correlated inversely with the number of nucleated cells infused. These data provide internally controlled in vivo evidence that, concomitantly with their proliferation, hematopoietic stem cells lose telomere length; it is possible that, as a result, their proliferative potential is reduced. These findings must be taken into account when developing new protocols in which few stem cells are used for bone marrow transplantation or for gene therapy.
Treatment of 3T3-L1 cells with 0.1-1.0 nM insulin results in rapid (5-15 min) activation of a soluble protein kinase that phosphorylates serine residues in ribosomal protein S6. The insulin-stimulated kinase activity is detectable in confluent, nongrowing preadipocytes and adipocytes. In the presence of 2 j.g of cycloheximide per ml, preconfluent 3T3-L1 cells also respond to insulin by acquiring an S6 kinase activity whose properties are the same as those of the enzyme activity elicited by insulin alone in growth-inhibited cells. The principal inslin-stimulated S6 kinase has a Mr of -50,001-0,000; there is a variable amount of activity that sediments with a Mr of about 80,000. The soluble enzyme exhibits optimal activity between pH 8 and pH 9, requires Mg2' (10-20 mM), and is inhibited by Ca2+ (0.5 mM), Mn2+ (0.05 mM), and NaF (30 mM). GTP cannot substitute for ATP in the phosphotransferase reaction; cAMP, cGMP, phosphatidylserine plus diolein, the cAMP-dependent protein kinase inhibitor, and heparin (0.7 Iug/ml) are without effect. Although treatment of 3T3-L1 cells with insulin does not influence the activity or the subcellular distribution of the phospholipid and Ca2+-dependent protein kinase C, exposure to the phorbol tumor promoter phorbol 12-myristate 13-acetate (PMA) results in translocation of protein kinase C to the membrane and activation of a soluble phospholipid and Ca2+-independent S6 protein kinase that has the same magnitude of activity and sedimentation behavior as the insulin-induced activity. Trypsin treatment of either 3T3-Li cytosolic extracts or partially purified 3T3-L1 protein kinase C generates a small amount of S6 kinase activity of Mr 50,000. This activity, resolved by sucrose gradient centrifugation, is less active than that elicited by either insulin or PMA and, unlike the activities generated by insulin and PMA, is associated with histone kinase activity. The data suggest that the S6 kinase elicited by either insulin or PMA is neither protein kinase C, its phospholipid, and Ca2+-independent proteolytic derivative nor the result of proteolytic activation of an inactive proenzyme that can be reproduced by trypsin treatment of cell extracts in vitro.Multisite phosphorylation of ribosomal protein S6 in cultured cells occurs in response to the addition of serum (1-3), hormones and growth factors (including insulin) (4-9), and phorbol esters (10,11). In vitro, S6 is a substrate for several different protein kinases, including cAMP-and cGMPdependent protein kinases (12-15), rat brain phospholipid and Ca2+-dependent kinase (16), lymphocyte H4 kinase (17), protease-activated kinases (PAK II) from reticulocytes (18), liver (19), and 3T3-L1 cells (5), and a membrane-associated protein kinase from 3T3-L1 cells (20). A dramatic increase in soluble S6 kinase activity has recently been reported to occur in serum-treated quiescent fibroblasts (3). This activation, measured in cell extracts, was stabilized by buffers containing EGTA and fB-glycerophosphate, suggesting the possibility that mod...
Core Facilities are key elements in the research portfolio of academic and private research institutions. Administrators overseeing core facilities (core administrators) require assessment tools for evaluating the need and effectiveness of these facilities at their institutions. This article discusses ways to promote best practices in core facilities as well as ways to evaluate their performance across 8 of the following categories: general management, research and technical staff, financial management, customer base and satisfaction, resource management, communications, institutional impact, and strategic planning. For each category, we provide lessons learned that we believe contribute to the effective and efficient overall management of core facilities. If done well, we believe that encouraging best practices and evaluating performance in core facilities will demonstrate and reinforce the importance of core facilities in the research and educational mission of institutions. It will also increase job satisfaction of those working in core facilities and improve the likelihood of sustainability of both facilities and personnel.
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