Low energy structural dynamics and constrained libration of Li(NH3)4, the lowest melting point metal.

The lattice and molecular dynamics for the solid phases of the lowest melting-point metal, Li(NH3)4, are determined by incoherent inelastic neutron scattering. Measurements of internal molecular displacements and distortions of the Li(NH3)4 units have been modelled and assigned using density functional theory calculations for the solid and molecular system. Inelastic neutron scattering measurement allow for the first determination of NH3 librational transitions.

The structure, thermal properties and phase transformations of the cubic polymorph of magnesium tetrahydroborate.

The structure of the cubic polymorph of magnesium tetrahydroborate (γ-Mg(BH(4))(2)) has been determined in space group Ia3d from a structural database of the isoelectronic compound SiO(2); this has been corroborated by DFT calculations. The structure is found to concur with that recently determined by Filinchuk et al. (Y. Filinchuk, B. Richter, T. R. Jensen, V. Dmitriev, D. Chernyshov and H. Hagemann, Angew. Chem. Int. Ed., 2011, DOI: 10.1002/anie.201100675). The phase transformations and subsequent decomposition of γ-Mg(BH(4))(2) on heating have been ascertained from variable-temperature synchrotron X-ray diffraction data combined with thermogravimetric and mass spectrometry measurements. At ~160 °C, conversion to a disordered variant of the ß-Mg(BH(4))(2) phase (denoted as ß') is observed along with a further unidentified polymorph. There is evidence of amorphous phases during decomposition but there is no direct crystallographic indication of the existence of Mg(B(12)H(12)) or other intermediate Mg-B-H compounds. MgH(2) and finally Mg are observed in the X-ray diffraction data after decomposition.

Pattern recognition receptor expression is not impaired in patients with chronic mucocutanous candidiasis with or without autoimmune polyendocrinopathy candidiasis ectodermal dystrophy.

Patients with chronic mucocutaneous candidiasis (CMC) have an unknown primary immune defect and are unable to clear infections with the yeast Candida. CMC includes patients with AIRE gene mutations who have autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), and patients without known mutations. CMC patients have dysregulated cytokine production, suggesting that defective expression of pattern recognition receptors (PRRs) may underlie disease pathogenesis. In 29 patients with CMC (13 with APECED) and controls, we assessed dendritic cell (DC) subsets and monocyte Toll-like receptor (TLR) expression in blood. We generated and stimulated monocyte-derived (mo)DCs with Candida albicans, TLR-2/6 ligand and lipopolysaccharide and assessed PRR mRNA expression by polymerase chain reaction [TLR-1-10, Dectin-1 and -2, spleen tyrosine kinase (Syk) and caspase recruitment domain (CARD) 9] in immature and mature moDCs. We demonstrate for the first time that CMC patients, with or without APECED, have normal blood levels of plasmocytoid and myeloid DCs and monocyte TLR-2/TLR-6 expression. We showed that in immature moDCs, expression levels of all PRRs involved in anti-Candida responses (TLR-1, -2, -4, -6, Dectin-1, Syk, CARD9) were comparable to controls, implying that defects in PRR expression are not responsible for the increased susceptibility to Candida infections seen in CMC patients. However, as opposed to healthy controls, both groups of CMC patients failed to down-regulate PRR mRNA expression in response to Candida, consistent with defective DC maturation, as we reported recently. Thus, impaired DC maturation and consequent altered regulation of PRR signalling pathways rather than defects in PRR expression may be responsible for inadequate Candida handling in CMC patients.

Impaired dendritic cell maturation and cytokine production in patients with chronic mucocutanous candidiasis with or without APECED.

Patients with chronic mucocutaneous candidiasis (CMC) suffer persistent infections with the yeast Candida. CMC includes patients with autoimmune regulator (AIRE) gene mutations who have autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), and patients without known mutations. CMC patients have dysregulated cytokine production, and dendritic cells (DCs), as central orchestrators, may underlie pathogenic disease mechanisms. In 29 patients with CMC (13 with APECED) and controls, we generated monocyte-derived DCs, stimulated them with Candida albicans, Toll-like receptor-2/6 ligand and lipopolysaccharide to assess cytokine production [interleukin (IL)-12p70, IL-23, interferon (IFN)-gamma, IL-2, tumour necrosis factor (TNF)-alpha, IL-6, transforming growth factor-beta, IL-10, IL-5, IL-13] and cell-surface maturation marker expression (CD83, CD86, human leucocyte antigen D-related). In both APECED and non-APECED CMC patients, we demonstrate impairment of DC function as evidenced by altered cytokine expression profiles and DC maturation/activation: (1) both groups over-produce IL-2, IFN-gamma, TNF-alpha and IL-13 and demonstrate impaired DC maturation. (2) Only non-APECED patients showed markedly decreased Candida-stimulated production of IL-23 and markedly increased production of IL-6, suggesting impairment of the IL-6/IL-23/T helper type 17 axis. (3) In contrast, only APECED patients showed DC hyperactivation, which may underlie altered T cell responsiveness, autoimmunity and impaired response to Candida. We demonstrate different pathogenic mechanisms on the same immune response pathway underlying increased susceptibility to Candida infection in these patients.

Proteasome inhibition elicits a biphasic effect on neuronal apoptosis via differential regulation of pro-survival and pro-apoptotic transcription factors.

The role of the proteasome in neuronal apoptosis is poorly understood since both anti- and pro-apoptotic effects result from proteasome inhibition. We studied the effects of proteasome inhibition in cultured rat cerebellar granule neurons. Acute exposure to proteasome inhibitors MG-132 and lactacystin blocked caspase activation induced by removal of depolarizing medium. However, chronic treatment with MG-132 activated caspases in neurons maintained in depolarizing potassium. The biphasic effect of MG-132 was hypothesized to be due to differential degradation of anti- and pro-apoptotic proteins. Accordingly, acute exposure to MG-132 inhibited the hyperphosphorylation, loss of DNA binding, ubiquitination, and degradation of the pro-survival transcription factor MEF2D induced by removal of depolarizing medium. In contrast, chronic exposure to MG-132 increased the expression and phosphorylation of c-Jun, elevated levels of the pro-apoptotic protein Bim, and triggered neuronal apoptosis, even in the presence of depolarizing medium. Thus, proteasome inhibition exerts an acute pro-survival action by stabilizing MEF2 transcription factors. However, chronic proteasome inhibition causes a build-up of phosphorylated c-Jun and Bim, which eventually overwhelms the effects of MEF2 and triggers apoptosis.

Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy.

Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.

Tim23p contains separate and distinct signals for targeting to mitochondria and insertion into the inner membrane.

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.

Characterization of the mitochondrial inner membrane translocase complex: the Tim23p hydrophobic domain interacts with Tim17p but not with other Tim23p molecules.

Tim23p is a mitochondrial inner membrane protein essential for the import of proteins from the cytosol. Tim23p contains an amino-terminal hydrophilic segment and a carboxyl-terminal hydrophobic domain (Tim23Cp). To study the functions and interactions of the two parts of Tim23p separately, we constructed tim23N, encoding only the hydrophilic region of Tim23p, and tim23C, encoding only the hydrophobic domain of Tim23p. Only the Tim23C protein is imported into mitochondria, indicating that the mitochondrial targeting information in Tim23p resides in its membrane spans or intervening loops. Tim23Cp, however, cannot substitute for full-length Tim23p, suggesting that the hydrophilic portion of Tim23p also performs an essential function in mitochondrial protein import. We found that overexpression of Tim23Cp is toxic to yeast cells that carry the tim23-1 mutation. Excess Tim23Cp causes Tim23-1p to disappear, leaving tim23-1 cells without a full-length version of the Tim23 protein. If Tim17p, another inner membrane import component, is overexpressed along with Tim23Cp, the toxicity of Tim23Cp is largely reversed and the Tim23-1 protein no longer disappears. In coimmunoprecipitations from solubilized mitochondria, Tim17p associates with the Tim23C protein. In addition, we show that Tim23p and Tim17p can be chemically cross-linked to each other in intact mitochondria. We conclude that the hydrophobic domain encoded by tim23C targets Tim23p to the mitochondria and mediates the direct interaction between Tim23p and Tim17p. In contrast, Tim23Cp cannot be coimmunoprecipitated with Tim23p, raising the possibility that the hydrophobic domain of Tim23p does not interact with other Tim23 molecules.

Persistence of peptide-induced CD4+ T cell anergy in vitro.

Clonal T cell unresponsiveness, or anergy, has been proposed as a mechanism of peripheral tolerance in vivo, and as a potential means of curbing unwanted T cell responses. In this study, anergy was induced in a T helper cell (Th) clone reactive to hemoglobin (Hb) peptide 64-76 by coculture of the T cells with live antigen-presenting cells (APCs) and 74L, a peptide analog of Hb(64-76) that contains a single amino acid substitution of leucine for glycine at position 74, or with a low concentration of the agonist ligand. The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64-76), and was not the result of impaired TCR/CD3 downmodulation. The addition of exogenous IL-12 transiently restored proliferation of the anergic lines, but removal of IL-12 from culture returned the T cells to their nonproliferative state. Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2. Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.

SMS1, a high-copy suppressor of the yeast mas6 mutant, encodes an essential inner membrane protein required for mitochondrial protein import.

MAS6 encodes an essential inner membrane protein required for mitochondrial protein import in the yeast Saccharomyces cerevisiae (Emtage and Jensen, 1993). To identify new inner membrane import components, we isolated a high-copy suppressor (SMS1) of the mas6-1 mutant. SMS1 encodes a 16.5-kDa protein that contains several potential membrane-spanning domains. The Sms1 protein is homologous to the carboxyl-terminal domain of the Mas6 protein. Like Mas6p, Sms1p is located in the mitochondrial inner membrane and is an essential protein. Depletion of Sms1p from cells causes defects in the import of several mitochondrial precursor proteins, suggesting that Sms1p is a new inner membrane import component. Our observations raise the possibility that Sms1p and Mas6p act together to translocate proteins across the inner membrane.

Mas6p can be cross-linked to an arrested precursor and interacts with other proteins during mitochondrial protein import.

Mas6p is an integral membrane protein of the yeast mitochondrial inner membrane, which is essential for mitochondrial protein import (1). To determine whether Mas6p is directly involved in recognizing precursors or translocating them across the inner membrane, we asked if Mas6p was in close proximity to precursor proteins being imported into mitochondria. We report here that Mas6p can be chemically cross-linked to an imported protein arrested in transit through the mitochondrial inner membrane. Antiserum to Mas6p specifically immunoprecipitates one of several different mitochondrial proteins that are cross-linked to blocked precursors. Our results strongly suggest that Mas6p physically interacts with precursors during their translocation into the matrix. In addition, at least two other mitochondrial proteins that are each cross-linked to arrested precursors can be coimmunoprecipitated along with Mas6p under non-denaturing conditions. These observations provide evidence for a complex of proteins including Mas6p, each of which interacts with mitochondrial precursors during import.

Medicaid fraud in New York State.

New York has taken a hard line on Medicaid fraud. Since its inception in 1975, the stat's Medicaid Fraud Control Unit has recovered more than $116 million in overpayments, fines, and restitution.

Estrogens and atherosclerosis.

PIP: Factors involved in the development of coronary atherosclerosis and the possible role of estrogens in its development are discussed. Risk factors in the development of atherosclerosis include hyperlipemia, hypertension, cigarette smoking, and diabetes. However, the incidence of heart disease and presence of risk factors are also related to heredity, geography, and socioeconomic conditions, and to diet, exercise, and emotional stress. Contrary to previous belief, high doses of estrogens aggravate the condition of men and menopausal women at risk of heart attack. Although estrogens do not markedly alter cholesterol levels, they do tend to elevate triglyceride levels and contribute to hyperlipemia. They are also associated with diabotegenic sequelae and hypertension. Pregnancy and estrogens increase blood clotting Factors VII and X, accelerate prothrombin time, shorten clotting time, and incre ase platelef aggregation. Further research into the role of estrogens in the development of atherosclerosis is recommended.^ieng