Inhibitory effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) on the astrocytic sodium responses to glutamate.

Astrocytes are responsible for the majority of the clearance of extracellular glutamate released during neuronal activity. dl-threo-beta-benzyloxyaspartate (TBOA) is extensively used as inhibitor of glutamate transport activity, but suffers from relatively low affinity for the transporter. Here, we characterized the effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), a recently developed inhibitor of the glutamate transporter on mouse cortical astrocytes in primary culture. The glial Na(+)-glutamate transport system is very efficient and its activation by glutamate causes rapid intracellular Na(+) concentration (Na(+)(i)) changes that enable real time monitoring of transporter activity. Na(+)(i) was monitored by fluorescence microscopy in single astrocytes using the fluorescent Na(+)-sensitive probe sodium-binding benzofuran isophtalate. When applied alone, TFB-TBOA, at a concentration of 1 muM, caused small alterations of Na(+)(i). TFB-TBOA inhibited the Na(+)(i) response evoked by 200 muM glutamate in a concentration-dependent manner with IC(50) value of 43+/-9 nM, as measured on the amplitude of the Na(+)(i) response. The maximum inhibition of glutamate-evoked Na(+)(i) increase by TFB-TBOA was >80%, but was only partly reversible. The residual response persisted in the presence of the AMPA/kainate receptor antagonist CNQX. TFB-TBOA also efficiently inhibited Na(+)(i) elevations caused by the application of d-aspartate, a transporter substrate that does not activate non-NMDA ionotropic receptors. TFB-TBOA was found not to influence the membrane properties of cultured cortical neurons recorded in whole-cell patch clamp. Thus, TFB-TBOA, with its high potency and its apparent lack of neuronal effects, appears to be one of the most useful pharmacological tools available so far for studying glial glutamate transporters.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

Correlations of glycogen synthase and phosphorylase activities with glycogen concentration in human muscle biopsies. Evidence for a double-feedback mechanism regulating glycogen synthesis and breakdown.

The purpose of this study was to verify in man the relationships of muscle glycogen synthase and phosphorylase activities with glycogen concentration that were reported in animal studies. The upper level of glycogen concentration in muscle is known to be tightly controlled, and glycogen concentration was reported to have an inhibitory effect on synthase activity and a stimulatory effect on phosphorylase activity. Glycogen synthase and phosphorylase activity and glycogen concentration were measured in muscle biopsies in a group of nine normal subjects after stimulating an increase of their muscle glycogen concentration through either an intravenous glucose-insulin infusion to stimulate glycogen synthesis, or an Intralipid (Vitrum, Stockholm, Sweden) infusion in the basal state to inhibit glycogen mobilization by favoring lipid oxidation at the expense of glucose oxidation. Phosphorylase activity increased from 71.3 +/- 21.0 to 152.8 +/- 20.0 nmol/min/mg protein (P < .005) after the glucose-insulin infusion. Phosphorylase activity was positively correlated with glycogen concentration (P = .005 and P = .0001) after the glucose-insulin and Intralipid infusions, respectively. Insulin-stimulated glycogen synthase activity was significantly negatively correlated with glycogen concentration at the end of the Intralipid infusion (P < .005). In conclusion, by demonstrating a negative correlation of glycogen concentration with glycogen synthase and a positive correlation with phosphorylase, this study might confirm in man the double-feedback mechanism by which changes in glycogen concentration regulate glycogen synthase and phosphorylase activities. It suggests that this mechanism might play an important role in the regulation of glucose storage.

Serum Leptin Concentration, Body Mass Index and Incident Heart Failure: The Health, Aging, and Body Composition Study

Background: Leptin is produced primarily by adipocytes. Although originally associated with the central regulation of satiety and energy metabolism, increasing evidence indicates that leptin may be an important factor for congestive heart faire (CHF). In the study, we aimed to test the hypothesis that leptin may influence CHF pathophysiology via a pathway of increasing body mass index (BMI). Methods: We studied 2,389 elderly participants aged 70 and older (M; 1161, F: 1228) without CHF and with serum leptin measures at the Health Aging, and Body Composition study. We analyzed the association between serum leptin level and risk of incident CHF using Cox hazard proportional regression models. Elevated leptin level was defined as more than the highest quartile (Q4) of leptin distribution in the total sample for each gender. Adjusted-covariates included demographic, behavior, lipid and inflammation variables (partially-adjusted models), and further included BMI (fully-adjusted models). Results: In a mean 9-year follow-up, 316 participants (13.2%) developed CHF. The partially-adjusted models indicated that men and women with elevated serum leptin levels (>=9.89 ng/ml in men and >=25 ng/ml in women) had significantly higher risks of developing CHF than those with leptin level of less than Q4. The adjusted hazard ratios (95%CI) for incident CHF was 1.49 (1.04 -2.13) in men and 1.71 (1.12 -2.58) in women. However, these associations became non-significant after adjustment for including BMI for each gender. The fully-adjusted hazard ratios (95%CI) were 1.43 (0.94 -2.18) in men and 1.24 (0.77-1.99) in women. Conclusion: Subjects with elevated leptin levels have a higher risk of CHF. The study supports the hypothesis that the influence of leptin level on risk of CHF may be through a pathway related to increasing BMI.

Chemical genetic analysis of cdc2p : new insights into the regulation of cytokinesis in "Schizosaccharomyces pombe"

RÉSUMÉ La protéine kinase cyciine-cdc2p (Cdk) joue un rôle fondamental dans la progression du cycle cellulaire dans la levure de fission Schizosaccharomyces pombe. Nous avons étudié le rôle de cdc2p dans la régulation de la cascade de septation ou SIN (septation initiation network) en mitose et en méiose. Le SIN contrôle l'initiation de la cytokinèse à la fin de la mitose, et est supposé être négativement régulé par cdc2p. Nous avons mutagénéisé le site actif de cdc2p afin qu'il puisse lier un analogue de l'ATP (PP1) qui agit comme inhibiteur. Cet analogue ne peut pas lier la kinase de type sauvage. Cette approche dite «chemical genetics» permet une meilleure résolution temporelle comparée à l'approche classique utilisant les mutants sensibles à une température élevée. Nous avons montré que ce mutant cdc2-as (analogue sensitive) est fonctionnel et que, in vitro, l'activité kinase est inhibée en présence de l'analogue. Les cellules portant cette mutation, contrairement aux cellules de type sauvage s'arrêtent de manière irréversible soit en G2 soit en G1 et G2, suivant la concentration de l'inhibiteur. L'inactivation de cdc2p-as dans des cellules arrêtées en métaphase conduit au recrutement asymétrique des protéines du SIN sur le pôle du fuseau mitotique et au recrutement des composants du SIN, ainsi que de la ß-(1,3)glucan synthase à l'anneau contractile. De plus, nos résultats montrent que l'orthologue de la phosphatase cdc14p dans S. pombe, fip1p/clp1p, joue un rôle dans la régulation de la localisation des protéines du SIN suite à l'inactivation de cdc2p. Finalement, l'activité de cdc2p est requise pour maintenir la polo-like kinase plo1p sur les pôles du fuseau mitotique dans les premiers stages de la mitose. C'est pourquoi nous concluons que l'inactivation de cdc2p est suffisante pour activer le SIN et promouvoir la cytokinèse. Dans une étude séparée, nous avons caractérisé des potentiellement nouveaux composants ou régulateurs du SIN qui ont été isolés dans deux criblages génétiques visant à isoler des mutants atténuants la signalisation du SIN. Summary : The cyclin dependent protein kinase (Cdk) cdc2p plays a central role in the cell cycle progression of fission yeast Schizosaccharomyces pombe. We have studied the role of cdc2p in regulating the septation initiation network (SIN) in mitosis and meiosis. The SIN regulates the initiation of cytokinesis at the end of mitosis and is thought to be inhibited by cdc2p. We have mutated the active site of cdc2p to permit binding of an inhibitory ATP analogue (PP1), which is unable to bind unmodified kinases. This "chemical genetic" approach provides a much higher temporal resolution than it can be achieved with classical temperature-sensitive mutants. We demonstrate that cdc2-as (analogue sensitive) is functional and that addition of PP1 inhibits cdc2p kinase activity in vitro. Cells carrying the cdc2-as allele, but not cdc2+, undergo reversible cell cycle arrest following addition of PP1 either in G2, or at both major commitment points in the cell cycle (G1 and G2), depending upon the concentration of PP1. Inactivation of cdc2p-as in cells arrested in early mitosis promotes both the asymmetric recruitment of SIN proteins to the spindle pole bodies (SPBs), and the recruitment of the most downstream SIN components and ß-(1,3)-glucan synthase to the contractile ring. Furthermore, our results indicate that the S. pombe orthologue of Cdc14p, flp1p/clp1p, plays a role in regulating the relocalisation of SIN proteins following inactivation of cdc2p, and that cdc2p activity is required to retain the polo like kinase plot p on the SPBs in early mitosis. Thus, we conclude that inactivation of cdc2p is sufficient to activate the SIN and to promote cytokinesis. In a separate study, we have initially characterised potential novel components or regulators of the SIN pathway identified by two genetic screens for mutants attenuating SIN signaling.

Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

Catabolite repression in Pseudomonas aeruginosa PAO1 regulates the uptake of C4 -dicarboxylates depending on succinate concentration.

In Pseudomonas aeruginosa carbon catabolite repression (CCR) is exerted by the CbrA/B-CrcZ-Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C4 -dicarboxylates, impairs the utilization of less preferred substrates. When non-preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non-preferred substrates. The C4 -dicarboxylate transport (Dct) system in P. aeruginosa is composed of two main transporters: DctA, more efficient at mM succinate concentrations, and DctPQM, more important at μM. In this study, we demonstrate that the Dct transporters are differentially regulated by Crc, depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post-transcriptionally. The activation of dctA is explained by a Crc-mediated repression of dctR, encoding a transcriptional repressor of dctA. At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM. As a result, expression of dctA is reduced and that of dctPQM is increased.

Large-scale imatinib dose-concentration-effect study in CML patients under routine care conditions.

This observational study analyzed imatinib pharmacokinetics and response in 2478 chronic myeloid leukemia (CML) patients. Data were obtained through centralized therapeutic drug monitoring (TDM) at median treatment duration of ≥2 years. First, individual initial trough concentrations under 400mg/day imatinib starting dose were estimated. Second, their correlation (C^min(400mg)) with reported treatment response was verified. Low imatinib levels were predicted in young male patients and those receiving P-gp/CYP3A4 inducers. These patients had also lower response rates (7% lower 18-months MMR in male, 17% lower 1-year CCyR in young patients, Kaplan-Meier estimates). Time-point independent multivariate regression confirmed a correlation of individual C^min(400mg) with response and adverse events. Possibly due to confounding factors (e.g. dose modifications, patient selection bias), the relationship seemed however flatter than previously reported from prospective controlled studies. Nonetheless, these observational results strongly suggest that a subgroup of patients could benefit from early dosage optimization assisted by TDM, because of lower imatinib concentrations and lower response rates.

Macrophage Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis.

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Monoamine oxidase A down-regulation contributes to high metanephrine concentration in pheochromocytoma.

CONTEXT: The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. OBJECTIVE: The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. DESIGN: A multicenter comparative study was conducted. STUDY PARTICIPANTS: The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. MAIN OUTCOME MEASURES: CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. RESULTS: At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. CONCLUSION: High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

Oxytocin enhances the inhibitory effects of diazepam in the rat central medial amygdala.

Oxytocin is a neuropeptide that can reduce neophobia and improve social affiliation. In vitro, oxytocin induces a massive release of GABA from neurons in the lateral division of the central amygdala which results in inhibition of a subpopulation of peripherally projecting neurons in the medial division of the central amygdala (CeM). Common anxiolytics, such as diazepam, act as allosteric modulators of GABA(A) receptors. Because oxytocin and diazepam act on GABAergic transmission, it is possible that oxytocin can potentiate the inhibitory effects of diazepam if both exert their pre, - respectively postsynaptic effects on the same inhibitory circuit in the central amygdala. We found that in CeM neurons in which diazepam increased the inhibitory postsynaptic current (IPSC) decay time, TGOT (a specific oxytocin receptor agonist) increased IPSC frequency. Combined application of diazepam and TGOT resulted in generation of IPSCs with increased frequency, decay times as well as amplitudes. While individual saturating concentrations of TGOT and diazepam each decreased spontaneous spiking frequency of CeM neurons to similar extent, co-application of the two was still able to cause a significantly larger decrease. These findings show that oxytocin and diazepam act on different components of the same GABAergic circuit in the central amygdala and that oxytocin can facilitate diazepam effects when used in combination. This raises the possibility that neuropeptides could be clinically used in combination with currently used anxiolytic treatments to improve their therapeutic efficacy.

Prognostic value of postoperative carcinoembryonic antigen concentration and extent of invasion of resection margins after hepatic resection for colorectal metastases.

OBJECTIVE: To evaluate the prognostic value of postoperative concentration of carcinoembryonic antigen (CEA) and extent of surgical margins after resection of liver metastases from colorectal cancer. DESIGN: Retrospective study. SETTING: Teaching hospital, Switzerland. SUBJECTS: 49 patients with hepatic metastases after primary colorectal cancer. INTERVENTIONS: Resection of hepatic metastases MAIN OUTCOME MEASURES: Assessment of prognostic value of variables by univariate and multivariate analysis. RESULTS: Median survival was 24 months (range 5-86 months). Resection margins were clear (> 1-cm) in 10, close (< 1-cm) in 25 and invaded in 9 patients. On univariate analysis, a postoperative concentration of CEA of <4ng/ml was correlated with prolonged survival (p < 0.001), but the width of the resection margin was not of prognostic importance. There was no correlation between width of resection margins and postoperative concentration of CEA (p = 0.5). On multivariate analysis, postoperative concentrations of CEA of 4 ng/ml or more were associated with increased risk of death (relative risk 7.3; 95% confidence interval (CI) 2.8-18.7, p < 0.001). CONCLUSION: Postoperative CEA offers better prognostic discrimination than the width of resection margins after resection of liver metastases from colorectal tumours. Some patients with invaded resection margins did survive for 3 years, but no patient did whose CEA concentration was 4 ng/ml or more. The definition of a potentially curative hepatic resection should include a postoperative CEA concentration of <4 ng/ml (within the reference range).

Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis.

Macrophages are essential effector cells of innate immunity that play a pivotal role in the recognition and elimination of invasive microorganisms. Mediators released by activated macrophages orchestrate innate and adaptive immune host responses. The cytokine macrophage migration inhibitory factor (MIF) is an integral mediator of the innate immune system. Monocytes and macrophages constitutively express large amounts of MIF, which is rapidly released after exposure to bacterial toxins and cytokines. MIF exerts potent proinflammatory activities and is an important cytokine of septic shock. Recent investigations of the mechanisms by which MIF regulates innate immune responses to endotoxin and gram-negative bacteria indicate that MIF acts by modulating the expression of Toll-like receptor 4, the signal-transducing molecule of the lipopolysaccharide receptor complex. Given its role in innate immune responses to bacterial infections, MIF is a novel target for therapeutic intervention in patients with septic shock.

Stochastic time-concentration activity models for cytotoxicity in 3D brain cell cultures.

BACKGROUND: In vitro aggregating brain cell cultures containing all types of brain cells have been shown to be useful for neurotoxicological investigations. The cultures are used for the detection of nervous system-specific effects of compounds by measuring multiple endpoints, including changes in enzyme activities. Concentration-dependent neurotoxicity is determined at several time points. METHODS: A Markov model was set up to describe the dynamics of brain cell populations exposed to potentially neurotoxic compounds. Brain cells were assumed to be either in a healthy or stressed state, with only stressed cells being susceptible to cell death. Cells may have switched between these states or died with concentration-dependent transition rates. Since cell numbers were not directly measurable, intracellular lactate dehydrogenase (LDH) activity was used as a surrogate. Assuming that changes in cell numbers are proportional to changes in intracellular LDH activity, stochastic enzyme activity models were derived. Maximum likelihood and least squares regression techniques were applied for estimation of the transition rates. Likelihood ratio tests were performed to test hypotheses about the transition rates. Simulation studies were used to investigate the performance of the transition rate estimators and to analyze the error rates of the likelihood ratio tests. The stochastic time-concentration activity model was applied to intracellular LDH activity measurements after 7 and 14 days of continuous exposure to propofol. The model describes transitions from healthy to stressed cells and from stressed cells to death. RESULTS: The model predicted that propofol would affect stressed cells more than healthy cells. Increasing propofol concentration from 10 to 100 μM reduced the mean waiting time for transition to the stressed state by 50%, from 14 to 7 days, whereas the mean duration to cellular death reduced more dramatically from 2.7 days to 6.5 hours. CONCLUSION: The proposed stochastic modeling approach can be used to discriminate between different biological hypotheses regarding the effect of a compound on the transition rates. The effects of different compounds on the transition rate estimates can be quantitatively compared. Data can be extrapolated at late measurement time points to investigate whether costs and time-consuming long-term experiments could possibly be eliminated.