Search

In this paper, the authors describe how team-based project management processes and metrics enable design teams at Intel to plan, execute and evaluate performance on complex projects, on a quarterly basis.

Objectives.To investigate the effects of soluble interleukin-1 (IL-1) type II receptor (sIL-1RII) on a number of clinical, biochemical and histological parameters in rabbit antigen-induced arthritis.Methods.Arthritis was induced by intra-articular injection of methylated bovine serum albumin (mBSA) into rabbits pre-sensitized to the same antigen. An initial i.v. bolus of sIL-1RII was administered, followed by s.c. mini-pump dosing for 14 days, starting at the time of the arthritis induction. Animals received vehicle (saline 500 μl + 5 μl/h), low-dose sIL-1RII (13.4 μg + 1.34 μg/h) or high-dose sIL-1RII (40.2 μg + 4.02 μg/h).Results.Marked, dose-related inhibition of joint diameter, plasma prostaglandin E2 (PGE2), and synovial fluid IL-1α and IL-1β concentrations were seen after administration of sIL-1RII. However, synovial fluid PGE2 concentrations and synovial fluid cell counts were not affected. A significant inhibitory effect was also seen histologically on soft-tissue swelling and joint damage with high-dose sIL-1RII.Conclusions.These results demonstrate that IL-1 plays an important role in the pathogenesis of rabbit antigen-induced arthritis, thus confirming it as an excellent animal model with respect to evaluating anti-cytokine therapies for rheumatoid arthritis.Keywords:Arthritis, Inflammation, IL-1α, IL-1β, IL-1ra, sIL-1RII, Rabbit, Histology.

Perregaux and Gabel (Perregaux, D., and Gabel, C. A. (1994) J. Biol. Chem. 269, 15195-15203) reported that potassium depletion of lipopolysaccharide-stimulated mouse macrophages induced by the potassium ionophore, nigericin, leads to the rapid release of mature interleukin-1beta (IL-1beta). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1beta occurred, in the presence of nigericin (4-16 microM). No effects on the release of tumor necrosis factor-alpha, IL-6, or proIL-1beta were seen. Addition of the irreversible interleukin-1beta-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1beta release and activation of p45 ICE precursor. The radicicol analog itself did not inhibit ICE, but markedly, and very rapidly depleted intracellular levels of 31-kDa proIL-1beta. By contrast, dexamethasone, cycloheximide, and the Na+/H+ antiporter inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, had no effect on nigericin-induced release of IL-1beta. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1beta is dependent upon activation of p45 ICE processing. So far, the mechanism by which reduced intracellular potassium ion concentration triggers p45 ICE processing is not known, but further investigation in this area could lead to the discovery of novel molecular targets whereby control of IL-1beta production might be effected.

The kinetics of insulin-stimulated autophosphorylation of specific tyrosines in the β subunit of the mouse insulin receptor and activation of receptor kinase-catalyzed phosphorylation of a model substrate were compared. The deduced amino acid sequence of the mouse proreceptor was determined to locate tyrosine-containing tryptic peptides. Receptor was first incubated with unlabeled ATP to occupy nonrelevant autophosphorylation sites, after which [32P]autophosphorylation at relevant sites and attendant activation of substrate phosphorylation were initiated with [γ-32P]ATP and insulin. Activation of substrate phosphorylation underwent an initial lag of 10-20 s during which there was substantial 32P-autophosphorylation of tryptic phosphopeptides p2 and p3, but not p1. Following the lag, incorporation of 32P into p1 and the activation of substrate phosphorylation increased abruptly and exhibited identical kinetics. The addition of substrate to the receptor prior to ATP inhibits insulin-stimulated autophosphorylation, and consequently substrate phosphorylation. Insulin-stimulated autophosphorylation of the receptor in the presence of substrate inhibited primarily the incorporation of 32P into p1 and drastically inhibited substrate phosphorylation. From Edman radiosequencing of 32P-labeled p1, p2, and p3 and the amino acid sequence of the mouse receptor, the location of each phosphopeptide within the β subunit was determined. Further characterization of these phosphopeptides revealed that p1 and p2 represent the triply and doubly phosphorylated forms, respectively, of the region within the tyrosine kinase domain containing tyrosines 1148, 1152, and 1153. The doubly phosphorylated forms contain phosphotyrosines either at positions 1148 and 1152/1153 or positions 1152 and 1153. These results indicate that insulin stimulates sequential autophosphorylation of tyrosines 1148, 1152 and 1153, and that the transition from the doubly to the triply phosphorylated forms is primarily responsible for the activation of substrate phosphorylation.

The 5' flanking region of the mouse insulin proreceptor gene was isolated, and the 5' boundary of the minimal promoter was mapped. Genomic clones encompassing greater than 30 kilobases of the gene contain the promoter and exons 1 and 2 interrupted by an approximately 20-kilobase intron at the codon for amino acid 7 of the alpha subunit. The nucleotide sequence of a 1.3-kilobase fragment containing 766 base pairs of the 5' flanking region and the entire first exon was determined. Two major transcription start sites were mapped by S1 nuclease analysis to sites located 469 and 424 nucleotides upstream from the initiation codon for translation. The 5' terminus of an insulin proreceptor cDNA, isolated from a mouse 3T3-L1 adipocyte cDNA library, corresponds to the 3'-most major start site of transcription. The 5' deletion mutants of the 5' flanking region of the proreceptor gene, linked upstream of the bacterial chloramphenicol acetyltransferase reporter gene, were transfected into 3T3-L1 preadipocytes and assayed for promoter activity. The 5' boundary of the minimal promoter, which directs unexpectedly high levels of reporter gene expression, maps to a region 22 base pairs upstream from the 3'-most major transcription start site.

Heterologous proteins can be expressed in Xenopus laevis oocytes by cytoplasmic microinjection of mRNA. To circumvent limitations inherent in this approach we investigate direct nuclear injection of strong viral expression vectors to drive transcription and subsequent translation of cDNAs encoding cytoplasmic, secreted, and plasma membrane proteins. After several viral promoters had been tested, the pMT2 vector was found to be a superior expression vector for X. laevis oocytes capable of directing expression of high levels of functional heterologous proteins. Typically the amount of protein derived from transcription-translation of the microinjected cDNA accounts for approximately 1% of total non-yolk protein. Moreover, the inefficiency usually associated with nuclear injections was overcome by coinjection of pMT2 driving expression of a secreted alkaline phosphatase as an internal control to select positive-expressing oocytes. Using this method, we have successfully expressed high levels of chloramphenicol acetyltransferase, the adipocyte-specific cytosolic 422(aP2) protein, and the membrane-associated glucose transporter GLUT1. The system described should be applicable to a wide variety of proteins for which cDNAs are available. Hence, the cumbersome and often inefficient in vitro synthesis of mRNA for studying ion channels, receptors, and transporters as well as for expression cloning in Xenopus oocytes should no longer be necessary.