Static and dynamic efficiency of irreversible health care investments under alternative payment rules.

The paper studies the incentive for providers to invest in new health care technologies under alternative payment systems, when the patients' benefits are uncertain. If the reimbursement by the purchaser includes both a variable (per patient) and a lump-sum component, efficiency can be ensured both in the timing of adoption (dynamic) and the intensity of use of the technology (static). If the second instrument is unavailable, a trade-off may emerge between static and dynamic efficiency. In this context, we also discuss how the regulator could use control of the level of uncertainty faced by the provider as an instrument to mitigate the trade-off between static and dynamic efficiency. Finally, we calibrate the model to study a specific technology and estimate the cost of a regulatory failure.

ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex.

The small GTPase ADP-ribosylation factor (ARF) regulates the structure and function of the Golgi complex through mechanisms that are understood only in part, and which include an ability to control the assembly of coat complexes and phospholipase D (PLD). Here we describe a new property of ARF, the ability to recruit phosphatidylinositol-4-OH kinase-beta and a still unidentified phosphatidylinositol-4-phosphate-5-OH kinase to the Golgi complex, resulting in a potent stimulation of synthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate; this ability is independent of its activities on coat proteins and PLD. Phosphatidylinositol-4-OH kinase-beta is required for the structural integrity of the Golgi complex: transfection of a dominant-negative mutant of the kinase markedly alters the organization of the organelle.

ADP ribosylation factor regulates spectrin binding to the Golgi complex.

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, betaI Sigma* spectrin) and ankyrin (AnkG119 and an approximately 195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the in vitro association of betaI Sigma* spectrin with a Golgi fraction, that the Golgi-associated betaI Sigma* spectrin contains epitopes characteristic of the betaI Sigma2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP2), and that ARF recruits betaI Sigma* spectrin by inducing increased PtdInsP2 levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP2. We postulate that a PH domain within betaI Sigma* Golgi spectrin binds PtdInsP2 and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin's membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle.

Molecular cloning, expression, and developmental characterization of the murine retinoblastoma-related gene Rb2/p130.

The product of the retinoblastoma-related human gene Rb2/p130 is highly homologous with the product of the retinoblastoma tumor suppressor gene (pRb) and Rb-related p107. this homology is shared mainly in the pocket domain, a region that seems to play a key role in the functions of these proteins. Here we report the molecular cloning and initial characterization of the cDNA encoding the murine homologue of the human Rb2/p130 gene product. The 4.8-kb cDNA encodes a protein of 1125 amino acids that shows 90% indentity to that of the human protein. The Rb2/p130 mRNA is found to be expressed in all of the adult mouse tissues examined, with the highest level being detected in kidney and skeletal muscle. For the protein characterization, we used a polyclonal antibody raised against the COOH terminus of the human Rb2/p130 protein that also recognizes the mouse protein. In developing mouse embryos, the Rb2/p130 protein is expressed as early as day 10 of gestation and reached a peak of expression around day 13 of gestation, implying a developmental regulation of the Rb2/p130 gene in murine ontogeny.

Type 2 phosphatidylinositol 4-kinase is recruited to CD4 in response to CD4 cross-linking.

CD4 serves as a cell-cell adhesion molecule, with specific affinity for class II MHC molecules, and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein. Phosphoinositide (PI)-3-kinase and 1-phosphatidylinositol (PtdIns)-4-kinase activities were previously found to associate with the CD4:p56lck complex, but the protein responsible for PtdIns 4-kinase activity was not identified. Here we demonstrate that the 53 kDa type 2 PtdIns 4-kinase associates with CD4 using a monoclonal antibody specific for this enzyme. We also show that an increase in PtdIns 4-kinase activity is due to recruitment of the type 2 PtdIns 4-kinase protein to the CD4:p56lck complex after cross-linking with anti-CD4.

Phosphatidylinositol 4,5-bisphosphate synthesis is required for activation of phospholipase D in U937 cells.

Phospholipase D (PLD) has been implicated in signal transduction and membrane traffic. We have previously shown that phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) stimulates in vitro partially purified brain membrane PLD activity, defining a novel function of PtdIns-4,5-P2 as a PLD cofactor. In the present study we extend these observations to permeabilized U937 cells. In these cells, the activation of PLD by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) is greatly potentiated by MgATP. We have utilized this experimental system to test the hypothesis that MgATP potentiates PLD activation by G proteins because it is required for PtdIns-4,5-P2 synthesis by phosphoinositide kinases. As expected, MgATP was absolutely required for maintaining elevated phosphatidylinositol 4-phosphate (PtdIns-4-P) and PtdIns-4,5-P2 levels in the permeabilized cells. In the presence of MgATP, GTP gamma S further elevated the levels of the phosphoinositides. The importance of PtdIns-4,5-P2 for PLD activation was examined by utilizing a specific inhibitory antibody directed against phosphatidylinositol 4-kinase (PtdIns 4-kinase), the enzyme responsible for the first step in the synthesis of PtdIns-4,5-P2. Anti-PtdIns 4-kinase completely inhibited PtdIns 4-kinase activity in vitro and reduced by 75-80% PtdIns-4-P and PtdIns-4,5-P2 levels in the permeabilized cells. In parallel, the anti-PtdIns 4-kinase fully inhibited the activation of PLD by GTP gamma S and caused a 60% inhibition of PLD activation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, indicating that elevated PtdIns-4,5-P2 levels are required for PLD activation. This conclusion is supported by the fact that neomycin, a high affinity ligand of PtdIns-4,5-P2, also blocked PLD activation. Furthermore, the activity of PLD in U937 cell lysate was stimulated by PtdIns-4,5-P2 in a dose-dependent manner. The current results indicate that PtdIns-4,5-P2 synthesis is required for PLD activation in permeabilized U937 cells and strongly support the proposed function of PtdIns-4,5-P2 as a cofactor for PLD. In addition, the results further establish PtdIns-4,5-P2 as a key component in the generation of second messengers via multiple pathways including phosphoinositide-phospholipase C, phosphoinositide 3-kinase and PLD.

Novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipase D.

The activation of phospholipase D (PLD) is a receptor-mediated event that has been implicated in signal transduction and membrane traffic in eukaryotic cells. Little is known about the biochemical and molecular properties of signal-activated PLDs, and none has been isolated. Here we report that phosphatidylinositol 4,5-bisphosphate (PIP2) potently stimulates brain membrane PLD activity in vitro in a highly specific manner. PIP2 increases 10-fold the maximal activity of a partially purified PLD with an EC50 of < 0.5 mol %. Other acidic phospholipids, including phosphatidylinositol 4-phosphate, phosphatidylinositol, phosphatidylserine, and phosphatidic acid, are completely or nearly ineffective. Neomycin, a high affinity ligand of PIP2, inhibits membrane-bound PLD but has no effect on the activity of a detergent-solubilized or partially purified enzyme. The addition of PIP2 restores the sensitivity of partially purified PLD to neomycin inhibition, indicating that neomycin blocks membrane PLD activity by binding to endogenous PIP2. These results define a novel function of PIP2 as a cofactor for brain membrane PLD and suggest that PIP2 synthesis and hydrolysis could be important determinants in regulating PLD action in signal transduction and membrane transport.

Mechanism underlying superantigen-induced clonal deletion of mature T lymphocytes.

We observed that peripheral T cells activated in vivo or in vitro by superantigens are susceptible to cell death when their antigen receptor is cross-linked with the appropriate anti-alpha beta TCR mAb. TCR ligation by mAbs specifically drove the T cell clonal deletion in both CD4+ and CD8+ cell subsets. An IL-2/IL-2R interaction seems to be a critical step in predisposing superantigen activated cells to death; in fact, in vivo IL-2R blockade reversed T cell deletion in superantigen plus anti-alpha beta TCR mAb treated mice. TCR ligation by mAbs also produced cell death of the relevant targets in in vitro IL-2 activated T cells. Surprisingly, no T cell deletion was demonstrable in IL-2 activated cells following staphylococcal enterotoxin B--TCR interaction, ruling out the possibility that superantigen in itself can induce cell death. Thus, while superantigen activation opens the cell death program, a subsequent TCR--antigen (self) interaction appears necessary to produce clonal deletion in mature T lymphocytes.

In vivo and in vitro death of mature T cells induced by separate signals to CD4 and alpha beta TCR.

To investigate whether a clonal deletion mechanism is responsible for the mature T cell tolerance that may be induced in vivo by TCR signal to anti-CD4 (H129.19 mAb) coated cells, we analyzed the T cell repertoire in anti-CD4 mAb treated BALB/c mice by flow cytometry following TCR signals through anti-alpha beta TCR mAb or SEB superantigen. Lymph nodes showed a strong reduction in the CD4+/CD8+ cell ratio, and a selective clonal loss of CD4+ V beta 8+ cells 4d following anti-alpha beta TCR or SEB injection, respectively. Following lymph node cell activation in a short-term in vitro assay with SEB or anti-V beta 8 mAb, a selective elimination of CD4+ V beta 8+ cells was again detected, and DNA fragmentation analysis disclosed a cell death by apoptosis. These findings suggest that TCR triggering transduces an apoptotic signal into CD4+ mAb saturated cells that in turn leads to specific holes in the mature T cell repertoire.