Catalytic Mechanism of Mammalian Adenylyl Cyclase: A Computational Investigation.

Adenylyl cyclase (AC) catalyzes the synthesis of cyclic AMP, an important intracellular regulatory molecule, from ATP. We propose a catalytic mechanism for class III mammalian AC based on density functional theory calculations. We employ a model of the AC active site derived from a crystal structure of mammalian AC activated by Gα·GTP and forskolin at separate allosteric sites.

CD200R1 and CD200 expression are regulated by PPAR-γ in activated glial cells.

The mechanisms that control microglial activation are of interest, since neuroinflammation, which involves reactive microglia, may be an additional target in the search for therapeutic strategies to treat neurodegenerative diseases. Neuron-microglia interaction through contact-dependent or independent mechanisms is involved in the regulation of the microglial phenotype in both physiological and pathological conditions. The interaction between CD200, which is mainly present in neurons but also in astrocytes, and CD200R1, which is mainly present in microglia, is one of the mechanisms involved in keeping the microglial proinflammatory phenotype under control in physiological conditions.

MD + QM correlations with tryptophan fluorescence spectral shifts and lifetimes.

Principles behind quenching of tryptophan (Trp) fluorescence are updated and extended in light of recent 100-ns and 1-μs molecular dynamics (MD) trajectories augmented with quantum mechanical (QM) calculations that consider electrostatic contributions to wavelength shifts and quenching. Four studies are summarized, including (1) new insight into the single exponential decay of NATA, (2) a study revealing how unsuspected rotamer transitions affect quenching of Trp when used as a probe of protein folding, (3) advances in understanding the origin of nonexponential decay from 100-ns simulations on 19 Trps in 16 proteins, and (4) the correlation of wavelength with lifetime for decay-associated spectra (DAS). Each study strongly reinforces the concept that-for Trp-electron transfer-based quenching is controlled much more by environment electrostatic factors affecting the charge transfer (CT) state energy than by distance dependence of electronic coupling.

CCAAT/enhancer binding protein δ regulates glial proinflammatory gene expression.

The transcription factor CCAAT/enhancer binding protein δ (C/EBPδ) is expressed in activated astrocytes and microglia and can regulate the expression of potentially detrimental proinflammatory genes. The objective of this study was to determine the role of C/EBPδ in glial activation. To this end, glial activation was analyzed in primary glial cultures and in the central nervous system from wild type and C/EBPδ(-/-) mice.

Inhibition of CD200R1 expression by C/EBP β in reactive microglial cells.

Background: In physiological conditions, it is postulated that neurons control microglial reactivity through a series of inhibitory mechanisms, involving either cell contact-dependent, soluble-factor-dependent or neurotransmitter-associated pathways. In the current study, we focus on CD200R1, a microglial receptor involved in one of these cell contact-dependent mechanisms. CD200R1 activation by its ligand, CD200 (mainly expressed by neurons in the central nervous system),is postulated to inhibit the pro-inflammatory phenotype of microglial cells, while alterations in CD200-CD200R1 signalling potentiate this phenotype.

Simulations of tryptophan fluorescence dynamics during folding of the villin headpiece.

Protein folding kinetics is commonly monitored by changes in tryptophan (Trp) fluorescence intensity. Considerable recent discussion has centered on whether the fluorescence of the single Trp in the much-studied, fast-folding villin headpiece C-terminal domain (HP35) accurately reflects folding kinetics, given the general view that quenching is by a histidine cation (His(+)) one turn away in an α-helix (helix III) that forms early in the folding process, according to published MD simulations. To help answer this question, we ran 1.

Tissue plasminogen activator induces microglial inflammation via a noncatalytic molecular mechanism involving activation of mitogen-activated protein kinases and Akt signaling pathways and AnnexinA2 and Galectin-1 receptors.

Inflammatory responses mediated by glial cells play a critical role in many pathological situations related to neurodegeneration such as Alzheimer's disease. Tissue plasminogen activator (tPA) is a serine protease which best-known function is fibrinolysis, but it is also involved in many other physiological and pathological events as microglial activation. Here, we found that tPA is required for Aβ-mediated microglial inflammatory response and tumor necrosis factor-α release.

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein β.

Background: Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation.

C/EBPβ expression in activated microglia in amyotrophic lateral sclerosis.

Neuroinflammation is thought to play a pathogenic role in many neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). In this study we demonstrate that the expression of nitric oxide (NO) synthase-2 (NOS2), and cyclooxygenase (COX)-2 induced by lipopolysaccharide (LPS) with interferon-γ is higher in microglial-enriched cultures from G93A-SOD1 mice, an ALS animal model, than from wild type mice. The levels of CCAAT/enhancer binding protein β (C/EBPβ), a transcription factor that regulates proinflammatory gene expression, are also upregulated in activated G93A-SOD1 microglial cells.

CCAAT/enhancer binding protein delta in microglial activation.

The transcription factor CCAAT/enhancer binding protein delta (C/EBP delta) regulates transcription of genes that play important roles in glial activation. Previous studies have shown the astroglial expression of C/EBP delta but the microglial expression of C/EBP delta remains virtually unexplored, with the exception of two microarray studies. In this report, using murine primary cultures and BV2 cells we clearly demonstrate that C/EBP delta is expressed by microglia and it is upregulated in microglial activation.

Upregulation of p21Cip1 in activated glial cells.

The cdk inhibitor p21(Cip1), also named p21(Cip1/Waf1), is intimately involved in coupling growth arrest to cellular differentiation in several cell types. p21(Cip1) is a multifunctional protein that might regulate cell-cycle progression at different levels. In a recent study, we found no differences in the rate of proliferation between glial cells from wild-type and p21(Cip1-/-) mice.

European study on orthopaedic status of haemophilia patients with inhibitors.

Development of inhibitors against factor VIII (FVIII) or factor IX (FIX) in haemophilia patients is one of the most serious complications of repeated exposure to replacement therapy and has major clinical and economic consequences. To evaluate the relationship between inhibitor status of haemophilia patients and their quality of life (QoL) and degree of arthropathy and to compare the orthopaedic status of patients with/without inhibitors. An observational, cross-sectional, case control study enrolling: group A (n = 38), males aged 14-35 years, with severe congenital haemophilia A or B who had inhibitors against FVIII/FIX >5 years; group B (n = 41), as group A, but aged 36-65 years and group C (n = 49), as group A, but without inhibitors.

Ab initio prediction of tryptophan fluorescence quenching by protein electric field enabled electron transfer.

We report quantum mechanical-molecular mechanical (QM-MM) predictions of fluorescence quantum yields for 20 tryptophans in 17 proteins, whose yields span the range from 0.01 to 0.3, using ab initio computed coupling matrix elements for photoinduced electron transfer from the 1La excited indole ring to a local backbone amide.

Distribution of Clostridium perfringens epsilon toxin in the brains of acutely intoxicated mice and its effect upon glial cells.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxaemia in livestock. The disease is principally manifested as severe and often fatal neurological disturbance. Oedema of several organs, including the brain, is also a clinical sign related to microvascular damage.

CCAAT/enhancer binding protein-alpha is down-regulated by toll-like receptor agonists in microglial cells.

The transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) can regulate the expression of important genes in the inflammatory response, but little is known about its role in glial activation. By using primary cortical murine glial cultures, we show that C/EBPalpha is expressed by microglial cells in vitro. Lipopolysaccharide (LPS) down-regulates C/EBPalpha mRNA at 2 hr and all C/EBPalpha protein isoforms at 4 hr.

Upregulation of CCAAT/enhancer binding protein beta in activated astrocytes and microglia.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPbeta in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPbeta expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPbeta in untreated mixed glial cultures.

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia.

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells.

Effects of epidural anaesthesia-analgesia on intravenous anaesthesia with propofol.

The main purpose of this study was to demonstrate that the use of epidural anaesthesia-analgesia reduces the amount of propofol necessary to maintain surgical anaesthesia in dogs during ovariohysterectomy. The study was carried out on 28 bitches undergoing ovariohysterectomy with general anaesthesia using an intravenous infusion of propofol. Dogs were allocated to one of two groups.

Absence of the cell cycle inhibitor p21Cip1 reduces LPS-induced NO release and activation of the transcription factor NF-kappaB in mixed glial cultures.

We have studied possible differences in glial activation between cells from wild-type and p21Cip1-/- mice. We compared the effect of serum mitogenic stimulation on proliferation rate and on the total number of glial cells after 7 days of culture. No differences between wild-type and p21Cip1-/- glial cells were observed.

Effects of beta-AP peptides on activation of the transcription factor NF-kappaB and in cell proliferation in glial cell cultures.

The effect of two beta amyloid peptides (Abeta 25/35 and Abeta 1/42) on the activation of the transcription factor kappaB (NF-kappaB) in pure astroglial, pure microglial and mixed glial cell cultures was compared by means of single or double immunofluorescence and Western blot techniques. We also studied the effect of both peptides in cell proliferation in mixed glial cultures and pure astrocytes. The Abeta 1/42 peptide induced the activation of NF-kappaB in all studied cell cultures and its effect was potentiated by interferon-gamma (IFN-gamma).

Inhibition of beta-amyloid toxicity by short peptides containing N-methyl amino acids.

Single N-methyl amino acid-containing peptides related to the central hydrophobic region beta16-20 (Lys-Leu-Val-Phe-Phe) of the beta-amyloid protein are able to reduce the cytotoxicity of natural beta1-42 in PC12 cell cultures. N-methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t-test < 0.01%) and are nontoxic in the same assay.

High-yield isolation of murine microglia by mild trypsinization.

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.

Transplacental transfer of propofol in pregnant ewes.

Propofol is an injectable anaesthetic that is currently used both in veterinary and human medicine for the induction and maintenance of anaesthesia. Although little is known about the pharmacokinetics of propofol in fetuses, it is widely used in obstetric procedures, particularly in caesarean section. This study determines the pharmacokinetics of propofol in pregnant ewes in the last third of pregnancy, and placental transfer and pharmacokinetics in fetuses after the administration of a 6 mg/kg intravenous (i.

Red cell adenylate kinase deficiency: molecular study of 3 new mutations (118G>A, 190G>A, and GAC deletion) associated with hereditary nonspherocytic hemolytic anemia.

We report here 2 patients with chronic nonspherocytic hemolytic anemia (CNSHA) and severe red blood cell (RBC) adenylate kinase (AK) deficiency. One of these patients, a boy of Spanish origin, exhibited a neonatal icterus and splenomegaly and required blood transfusions until the age of 2 years. The other patient was a white, American infant born to parents who were first cousins; he also presented with neonatal icterus and anemia.

Structural, kinetic and cytotoxicity aspects of 12-28 beta-amyloid protein fragment: a reappraisal.

A chemical, structural and biological study on the beta-amyloid peptide beta12-28 is reported which was carried out in order to assess the feasibility using this peptide fragment as a model of the natural beta-amyloid protein. The aggregation properties of beta12-28 have been investigated by pulse field-gradient NMR spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. The results obtained suggest that beta12-28 behaviour is comparable to that of the natural beta-amyloid protein although kinetically slower.