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1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)13,14-dihydro-15-keto PGD(2)15-deoxy-Delta(12,14)-PGJ(2)PGJ(2)Delta(12)-PGJ(2)15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)PGJ(2)Delta(12)-PGJ(2)15-deoxy-Delta(12,14)-PGJ(2)13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.

In Huntington disease, polyglutamine expansion of the protein huntingtin (Htt) leads to selective neurodegenerative loss of medium spiny neurons throughout the striatum by an unknown apoptotic mechanism. Binding of Hip-1, a protein normally associated with Htt, is reduced by polyglutamine expansion. Free Hip-1 binds to a hitherto unknown polypeptide, Hippi (Hip-1 protein interactor), which has partial sequence homology to Hip-1 and similar tissue and subcellular distribution. The availability of free Hip-1 is modulated by polyglutamine length within Htt, with disease-associated polyglutamine expansion favouring the formation of pro-apoptotic Hippi-Hip-1 heterodimers. This heterodimer can recruit procaspase-8 into a complex of Hippi, Hip-1 and procaspase-8, and launch apoptosis through components of the 'extrinsic' cell-death pathway. We propose that Htt polyglutamine expansion liberates Hip-1 so that it can form a caspase-8 recruitment complex with Hippi. This novel non-receptor-mediated pathway for activating caspase-8 might contribute to neuronal death in Huntington disease.

The nucleation of the first-order phase transition of superfluid 3He-B from superfluid 3He-A is quite remarkable since it requires a seed of the order of a micron. We have studied this nucleation for 3He confined to a very dilute silica aerogel. This dirty superfluid behaves in a manner similar to previous reports for the pure superfluid. But we have discovered a novel magnetically driven nucleation switch acting on the pure superfluid- B phase. Last, we find the surprising result that the proximity effect between the pure and dirty superfluids at their interface is insufficient to nucleate the B phase in either superfluid.

Reasoned and defensible coverage decisions are essential for a fairer and more efficient healthcare system. Because healthcare resources are finite, coverage decisions should be informed by economic evaluations and made from a perspective that attends to the interests of both individuals and the population enrolled in a plan as a whole. Coverage decisions for all healthcare interventions should follow a 2-step procedure that consists of (1) the relatively impartial and objective assessment of an intervention's eligibility for coverage and (2) the distinctively value-laden determination (for which the enrolled population's values and preferences should take priority) to cover, conditionally cover, or not cover an intervention.

CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56(lck), a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56(lck). The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56(lck) could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56(lck). Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56(lck). Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.

The ground-state band of the Z=102 isotope 254No has been identified up to spin 14, indicating that the nucleus is deformed. The deduced quadrupole deformation, β=0.27, is in agreement with theoretical predictions. These observations confirm that the shell-correction energy responsible for the stability of transfermium nuclei is partly derived from deformation. The survival of 254No up to spin 14 means that its fission barrier persists at least up to that spin.

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.

The activity of Src-related protein-tyrosine kinases is repressed by the phosphorylation of a conserved carboxyl-terminal tyrosine by another cytoplasmic protein-tyrosine kinase termed p50csk. In this study, we characterize Ntk, a protein-tyrosine kinase bearing striking similarities to p50csk. Like p50csk, Ntk possesses Src homology 3 and Src homology 2 domains and lacks the consensus tyrosine phosphorylation and myristoylation sites found in members of the Src family. Expression of ntk transcripts was maximal in brain, and was observed at significant levels in thymus and spleen. ntk RNA levels were dramatically reduced upon mitogenic stimulation of normal T lymphocytes and were minimal in transformed T-cell populations. Firm evidence that Ntk is a Csk-related enzyme was provided by the observation that it phosphorylated a Src-related polypeptide on the inhibitory carboxyl-terminal tyrosine. These findings indicate that Ntk is a Csk-related enzyme that may play an inhibitory role in the control of T-cell proliferation.

Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.

The bill to restore federal funding for human fetal tissue research has been passed by the House and awaits Senate approval. But it requires women who are willing to donate fetal tissue to certify that they did not have an abortion with the intent to donate. It further requires researchers to keep the certifications on file and available for government audit. Both requirements spell trouble.H.R. 2507 passed by the US House of Representatives, is an attempt to end the ban on fetal tissue research. However it signals a new era in government violation of privacy and ultimately will undermine its own stated purpose. The law provides that any woman wishing to donate the fetal tissue from an abortion must sign 2 ethical intent forms. The 1st stating that the decision to donate was made independently of the decision to abort. The 2nd stating that the reason for aborting was not to donate fetal tissue. These forms of ethical intent are to be kept on file by the researchers using the fetal tissue and are to be made available for audit to ensure compliance. The law does not have any provision for protecting the confidentiality of these forms, nor does it state whose property they become. It also does not state who allowed to audit the records or whose responsibility it is. The results of this law are that women fearing exposure of their name in a document open to public audit will not want to donate. Further, in many states this failure to sign the forms can be legally viewed as intent to abort for donation and thus the abortion could be denied. In addition, researchers will not want to cover the expense or controversy surrounding these records and as such will refuse to accept fetal tissue.

The use of fetal tissue in transplants for treating illnesses such as Parkinson's disease and juvenile diabetes has raised the hopes of patients, their families, and the biomedical community. But, this practice has created considerable controversy. Concerns arise because tissue is usually obtained from electively aborted fetuses. Despite the controversy, there has been little systematic and sustained examination of the ethical and policy issues posed by the use of fetal tissue in biomedicine. The lack of information and analysis hampers serious discussion. In the Spring of 1988, the Center for Biomedical Ethics began an interdisciplinary research project on the scientific, ethical, and policy issues raised by the use of fetal tissue in biomedicine. Twenty-five scholars, drawn mainly, but not exclusively, from the faculty of the University of Minnesota, met to undertake the study. The members of this research group included experts in neonatology, pediatrics, neurology, neurosurgery, organ transplantation, tissue procurement, cell biology, immunology, epidemiology, law, philosophy, moral theology, and the behavioral sciences. The group met every three weeks over a period of ten months to collect and review information about the use of fetal tissue -- with special attention to transplantation -- the potential sources of fetal tissue, and the relevant laws and guidelines in the U.S. and other nations. Six members of the research group had primary responsibility for writing this report.

To the Editor. —Health plans customarily have excluded coverage for investigational interventions and covered most proven interventions. Although the distinction between investigational and proven has been pivotal, it remains blurry and notoriously unhelpful. The lack of an agreed-on definition of investigational , for example, has been the source of innumerable legal challenges brought—usually successfully—by patients, often of coverage denials for investigational interventions. While Dr Eddy's 1 proposed process is the clearest yet for making defensible judgments about which interventions are investigational, it ultimately may not help health plans make better coverage decisions. The problem goes beyond the term investigational to the distinction itself: even if it could be unambiguously drawn via precise agreed-on definitions and criteria, the investigational vs proven distinction would be an inadequate framework for making coverage decisions. Coverage decisions no longer are, nor should they be, based on a judgment regarding the investigational status of an intervention. Many investigational interventions are

The bill to restore federal funding for human fetal tissue research has been passed by the House and awaits Senate approval. But it requires women who are willing to donate fetal tissue to certify that they did not have an abortion with the intent to donate. It further requires researchers to keep the certifications on file and available for government audit. Both requirements spell trouble.

We report on an intense source of cold and ultracold neutrons at the high-flux reactor at Grenoble: a metal guide conducts neutrons with wavelengths >20 A from a liquid deutrium cold source to a neutron turbine serving as a generator of a strong and wide beam of ultracold neutrons.

The amyloid-beta precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-beta (A beta) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimer's disease brains and colocalization of its APP cleavage product with A beta in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for A beta peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimer's disease.

Introduction Whole blood functional assays are pharmacologically relevant in the drug discovery process to evaluate potency in a relevant biological matrix, to support establishment of PK/PD relationships and to aid in human dose predictions. However development of B cell activation assays by BCR ligation in rat whole blood has not been previously described. The aim of the present study was to develop novel methods of B cell activation in rat whole blood. Methods B cell activation in rat whole blood was evaluated by measuring CD86 up-regulation via flow cytometry. Rat B cells in whole blood were stimulated with dextran-coupled anti-IgD or a combination of anti-IgD and TLR9 agonist. BTK, SYK, and PI3Kδ inhibitors were added to rat whole blood prior to activation with dextran-coupled anti-IgD or anti-IgD and TLR9 agonist combination for pharmacological validation of the assay. Results Both methods of stimulation in rat whole blood evoked robust B cell activation in a uni-modal fashion. Highly selective inhibitors of BTK, SYK, and PI3Kδ dose-dependently attenuated B cell activity evoked by both dextran-coupled anti-IgD and combined anti-IgD and TLR9 agonist. Compound potencies and rank order determined by the two assays were comparable. Discussion Two novel methods were developed to stimulate B cells in rat whole blood, that have the potential to be used to support drug discovery efforts in the therapeutic targeting of B cells. Furthermore, we pharmacologically validated these whole blood assays using highly selective inhibitors of BTK, SYK, and PI3Kδ, signaling kinases which are downstream of the B cell receptor.

Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this antigen after direct application to the lungs of Brown Norway rats.Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats.We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production.Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.

The authors report five cases of fertile women who have Karyotype XO. After a short review of the classification of Turner's syndrome and of the physiopathology of the defective gonads in these patients, 79 cases in the literature and these 5 cases were reviewed and they allow us: a precise description of the morphology of these patients, which is dominated by the high incidence of small growth and the presence of the various degrees of malformations often found in Turner's syndrome; to study the gonad structure and function of these patients who have spontaneous menstruation. And finally to analyse the prognosis of pregnancies that have occurred in 33 women with an XO clone. These women have not only a raised incidence of spontaneous abortions at 32% but also frequent fetal malformations and chromosome abnormalities in the products of conception. It is therefore vital to carry out early amniocentesis in these patients.

INVESTIGATIONS into the presence of histocompatibility antigens in mouse brain have given apparently conflicting results. Haemagglutinin1,2 and cytotoxin3 inhibition studies have revealed that the brain has an extremely small capacity for absorbing H-2 antibodies, compared with lymphoid tissues. Brain tissue, however, induces transplantation immunity4 and histocompatibility antigens are detected in neurones of the cerebral cortex by immuno-fluorescence5. The selective localization of the specific fluorescence to the neurones can explain these conflicting results, for the number of stained cells represents only a small proportion of the total brain tissue. This will be reflected by a very low absorptive capacity in quantitative inhibition studies.