Evaluation of total purchasing pilots in England and Scotland and implications for primary care groups in England: personal interviews and analysis of routine data

Objectives: To evaluate the reported achievements of the 52 first wave total purchasing pilot schemes in 1996-7 and the factors associated with these; and to consider the implications of these findings for the development of the proposed primary care groups.

Definition of amide protection factors for early kinetic intermediates in protein folding

Hydrogen–deuterium exchange experiments have been used previously to investigate the structures of well defined states of a given protein. These include the native state, the unfolded state, and any intermediates that can be stably populated at equilibrium. More recently, the hydrogen–deuterium exchange technique has been applied in kinetic labeling experiments to probe the structures of transiently formed intermediates on the kinetic folding pathway of a given protein. From these equilibrium and nonequilibrium studies, protection factors are usually obtained. These protection factors are defined as the ratio of the rate of exchange of a given backbone amide when it is in a fully solvent-exposed state (usually obtained from model peptides) to the rate of exchange of that amide in some state of the protein or in some intermediate on the folding pathway of the protein. This definition is straightforward for the case of equilibrium studies; however, it is less clear-cut for the case of transient kinetic intermediates. To clarify the concept for the case of burst-phase intermediates, we have introduced and mathematically defined two different types of protection factors: one is Pstruc, which is more related to the structure of the intermediate, and the other is Papp, which is more related to the stability of the intermediate. Kinetic hydrogen–deuterium exchange data from disulfide-intact ribonuclease A and from cytochrome c are discussed to explain the use and implications of these two definitions.

Sustained in vivo cardiac protection by a rationally designed peptide that causes ɛ protein kinase C translocation

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). ɛ Protein kinase C (ɛPKC) has been suggested to mediate preconditioning. Here, we describe an ɛPKC-selective agonist octapeptide, ψɛ receptor for activated C-kinase (ψɛRACK), derived from an ɛPKC sequence homologous to its anchoring protein, ɛRACK. Introduction of ψɛRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased ɛPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that ɛPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this ɛPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0–10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90–240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor α receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor α (TGFα) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFα cleavage 120–180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFα. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFα. Neutralization of TGFα function by an anti-TGFα antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFα–EGFR–MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge

IL-12 plays a central role in both the induction and magnitude of a primary Th1 response. A critical question in designing vaccines for diseases requiring Th1 immunity such as Mycobacterium tuberculosis and Leishmania major is the requirements to sustain memory/effector Th1 cells in vivo. This report examines the role of IL-12 and antigen in sustaining Th1 responses sufficient for protective immunity to L. major after vaccination with LACK protein (LP) plus rIL-12 and LACK DNA. It shows that, after initial vaccination with LP plus rIL-12, supplemental boosting with either LP or rIL-12 is necessary but not sufficient to fully sustain long-term Th1 immunity. Moreover, endogenous IL-12 is also shown to be required for the induction, maintenance, and effector phase of the Th1 response after LACK DNA vaccination. Finally, IL-12 is required to sustain Th1 cells and control parasite growth in susceptible and resistant strains of mice during primary and secondary infection. Taken together, these data show that IL-12 is essential to sustain a sufficient number of memory/effector Th1 cells generated in vivo to mediate long-term protection to an intracellular pathogen.

Protection from superoxide damage associated with an increased level of the YggX protein in Salmonella enterica

The deleterious effect of superoxide radicals on cell growth and survival is predominately caused by rapid oxidation of labile [Fe-S] clusters in proteins. Oxidation of these clusters releases Fe(II) ions, which participate in Fenton chemistry that damages DNA. Here it is shown that elevated levels of the YggX protein increase the resistance of Salmonella enterica to superoxide stress, reverse enzymatic defects attributed to oxidized [Fe-S] clusters, and decrease the spontaneous mutation frequency. The data are consistent with a model in which YggX protects protein [Fe-S] clusters from oxidation.