Immunohistochemical localization of dopamine in the brain of the insect Locusta migratoria migratorioides in comparison with the catecholamine distribution determined by the histofluorescence technique

Summary As part of a follow-up study to our previous investigation of the catecholaminergic neurosecretory cells in the brain of adult female locusts (Locusta migratoria migratorioides) by means of the formaldehyde-induced fluorescence method, we have attempted to specify the identity of the amines present in these cells by an immunohistological technique. Using a recently developed anti-dopamine serum, we have demonstrated that the majority of the cate cholaminergic median neurosecretory cells contain dopamine. Moreover, dopamine is present in some cell bodies of other zones of the brain, i.e. the median subocellar neurosecretory cells, perikarya in external areas of the protocerebrum, below the calyces, around the pedunculus, in the optic lobes (between the lobula and the medulla, between the medulla and the lamina), and in external zones of the tritocerebrum. Among the structured neuropils, which were particularly fluorescent in the formaldehyde-induced fluorescence method, only the pedunculus, the posterior part of the central body, the external zones of the - and lobes and the proximal part of the lamina contain little dopamine.     

Fibrates but not statins increase plasma selenium in dyslipidemic aged patients – The EVA study

This secondary analysis of “Etude du Vieillissement Artériel” (EVA) study reports the effect of fibrates and statins on plasma selenium concentration and its 9-year change in free-living dyslipidemic elderly. Dyslipidemic patients were categorized in three sub-groups according to final low-density lipoprotein (LDL)-cholesterol level or hypolipidemic treatment: non-treated dyslipidemic (LDL-cholesterol >4.41 mmol/L, n=84); dyslipidemics who were treated exclusively by fibrates (n=47) or by statins (n=25) whatever their serum LDL-cholesterol concentration. The influence of lipid-lowering treatments on plasma selenium concentrations and its 9-year change was evaluated by analysis of variance (ANOVA) and multivariate linear regression models taking into account cardiovascular risk and changes in lipid-profile parameters. Multivariate linear regression indicated that the plasma selenium decline was associated with the longitudinal variation in LDL (β=?0.039±0.019, p=0.04) and high-density lipoprotein (HDL)-cholesterol concentrations (β=0.187±0.059, p=0.002) but not with triglycerides (β=?0.018±0.031, p=0.57). During the 9-year follow-up, similar plasma selenium declines were observed in all the sub-groups (p=0.33) despite plasma selenium levels being higher in fibrate users and lower in statin users (p=0.0004). The mechanisms underlying these data are not yet totally understood, but considering the risk of selenium deficiency in the elderly and its relationship with poor health status further clinical trial is needed to verify the proposed hypotheses.     

High prevalence of cytomegalovirus (CMV) infection in infants born to HIV infected mothers–ANRS French Perinatal Cohort (EPF)

Whenever there is a sensational criminal case involving HIV transmission, the media cover it with far more gusto than they usually devote to scientific advances in the field. For example, a murder trial is now taking place in Canada involving a man who has been accused of sexually transmitting HIV to 11 different women, two of whom have died of their infections. Moreover, it is alleged that the accused perpetrator deliberately withheld from these women the fact that he was HIV-positive and that he refused to use a condom during intercourse. Notwithstanding that the suspect is possibly psychopathic and uncaring, or possibly of low intelligence and unable to assess the consequence of his actions, most people probably hope that he is convicted, sentenced, and imprisoned for his acts. Furthermore, most people probably wish for the criminal justice system to pursue these cases with vigour. In fact, however, people should understand that such legal action, and the willingness of the courts to hear these cases, will only weaken the global battle against HIV transmission.     

IL-10 and Lymphotoxin-α Expression Profiles within Marginal Zone-Like B-Cell Populations Are Associated with Control of HIV-1 Disease Progression

Understanding how the immune system facilitates or controls HIV-1 disease progression has important implications for the design of effective interventions. We report that although B-cell dysregulations associated with HIV-1 disease progression are accompanied by an overall decrease in the percentage of total blood B-cells, we observe an increase in relative frequencies of cells presenting characteristics of both transitional immature and first-line marginal zone (MZ) B-cell populations, we designated as precursor MZ-like B-cells. B-cells with similar attributes have been associated with IL-10 expression and “regulatory” potential. As such, the relative frequencies of precursor MZ-like B-cells expressing IL-10 are increased in the blood of viremic HIV-1-infected individuals when compared to HIV-negative subjects. Importantly, in aviremic HIV-1 Elite-Controllers (EC), we found unaltered relative percentages of precursor MZ-like B-cells which presented normal IL-10 expression patterns. Furthermore, EC had increased relative frequencies of blood MZ-like B-cells expressing LT-α. Thus in contrast to viremic HIV-1-infected individuals, EC present MZ-like B-cell populations which IL-10 and LT-α expression profiles may favour homeostasis of immune responses and lymphoid microenvironments.     

XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase β, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.The genomic integrity of cells is controlled by a network of protein factors that assess the status of the genome and either cause progression of proliferation or induce a halt in the cell cycle. In eukaryotes, DNA strand breaks, introduced either directly by ionizing radiation or indirectly following enzymatic incision of a DNA lesion, trigger the synthesis of poly(ADP-ribose) by the enzyme poly(ADP-ribose) polymerase (PARP) (1, 13, 39). At the site of breakage, PARP catalyzes the transfer of the ADP-ribose moiety from its substrate, NAD⁺, to a limited number of protein acceptors involved in chromatin architecture and DNA metabolism, including the enzyme itself. These modified proteins, which carry long chains of negatively charged ADP-ribose polymers, lose their affinity for DNA and are thus inactivated. The short half-life of the polymer is attributed to the high activity of poly(ADP-ribose) glycohydrolase, which cleaves the ribose-ribose bond (28, 30). Therefore, poly(ADP-ribosylation) is an immediate but transient postranslational modification of nuclear proteins, induced by DNA-damaging agents.The physiological role of PARP has been much debated in the last decade, but recent molecular and genetic approaches, including expression of either a dominant-negative mutant (26, 36, 44) or antisense oligonucleotides (14), have clearly implicated PARP in the base excision repair (BER) pathway. A more definitive assessment of PARP function was recently provided by the generation of PARP-deficient mice by homologous recombination (35, 53). We found that PARP^−/− mice are hypersensitive to monofunctional alkylating agents and γ-irradiation and display a marked genomic instability (sister chromatid exchanges and chromatid and chromosome breaks) following DNA damage (35). Interestingly, γ-irradiation of these mice causes acute toxicity of the epithelia of their small intestines (35), as has been observed with other DNA damage and signalling and repair enzyme deficiencies (2, 3), thus emphasizing the crucial function of DNA surveillance programs of rapidly dividing cells. Similar results indicating that PARP is important for the maintenance of genomic stability following environmental or experimental stress were recently obtained (54).In this work, we have used the two-hybrid system to identify genes encoding proteins that putatively interact with PARP and are involved in its biological function. The human PARP cDNA fused to the LexA-encoding DNA-binding domain (DBD) was used as bait to screen a HeLa cDNA library fused with the activation domain of Gal4. This screening resulted in the identification of the BER pathway protein XRCC1 (X-ray repair cross-complementing 1) as a factor that associates with PARP. This interaction was further confirmed by in vivo experiments with glutathione S-transferase (GST)-tagged fusion proteins expressed in Cos-7 and HeLa cells. XRCC1 and PARP were found to interact via their respective BRCT (BRCA1 C terminus) modules (4, 9) and via an additional site located in the N-terminal zinc-finger domain of PARP. This association dramatically decreased the catalytic activity of PARP without modifying its nick sensor function. Therefore, the association of PARP with XRCC1, a partner of DNA ligase III (7, 8) and DNA polymerase β (25), is suggestive of a role in the detection and protection of a DNA strand break and the subsequent targeting of a BER complex to the damaged site.     

Spatial variation in the management and outcomes of acute coronary syndrome

Background

Aprotinin has been shown to be effective in reducing peri-operative blood loss and the need for re-operation due to continued bleeding in cardiac surgery. The lysine analogues tranexamic acid (TXA) and epsilon aminocaproic acid (EACA) are cheaper, but it is not known if they are as effective as aprotinin.

Methods

Studies were identified by searching electronic databases and bibliographies of published articles. Data from head-to-head trials were pooled using a conventional (Cochrane) meta-analytic approach and a Bayesian approach which estimated the posterior probability of TXA and EACA being equivalent to aprotinin; we used as a non-inferiority boundary a 20% increase in the rates of transfusion or re-operation because of bleeding.

Results

Peri-operative blood loss was significantly greater with TXA and EACA than with aprotinin: weighted mean differences were 106 mls (95% CI 37 to 227 mls) and 185 mls (95% CI 134 to 235 mls) respectively. The pooled relative risks (RR) of receiving an allogeneic red blood cell (RBC) transfusion with TXA and EACA, compared with aprotinin, were 1.08 (95% CI 0.88 to 1.32) and 1.14 (95% CI 0.84 to 1.55) respectively. The equivalent Bayesian posterior mean relative risks were 1.15 (95% Bayesian Credible Interval [BCI] 0.90 to 1.68) and 1.21 (95% BCI 0.79 to 1.82) respectively. For transfusion, using a 20% non-inferiority boundary, the posterior probabilities of TXA and EACA being non-inferior to aprotinin were 0.82 and 0.76 respectively. For re-operation the Cochrane RR for TXA vs. aprotinin was 0.98 (95% CI 0.51 to 1.88), compared with a posterior mean Bayesian RR of 0.63 (95% BCI 0.16 to 1.46). The posterior probability of TXA being non-inferior to aprotinin was 0.92, but this was sensitive to the inclusion of one small trial.

Conclusion

The available data are conflicting regarding the equivalence of lysine analogues and aprotinin in reducing peri-operative bleeding, transfusion and the need for re-operation. Decisions are sensitive to the choice of clinical outcome and non-inferiority boundary. The data are an uncertain basis for replacing aprotinin with the cheaper lysine analogues in clinical practice. Progress has been hampered by small trials and failure to study clinically relevant outcomes.     

Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?

High fat intake is associated with fat mass gain through fatty acid activation of peroxisome proliferator-activated receptors delta and gamma, which promote adipogenesis. We show herein that, compared to a combination of specific agonists to both receptors or to saturated, monounsaturated, and omega-3 polyunsaturated fatty acids, arachidonic acid (C20:4, omega-6) promoted substantially the differentiation of clonal preadipocytes. This effect was blocked by cyclooxygenase inhibitors and mimicked by carbacyclin, suggesting a role for the prostacyclin receptor and activation of the cyclic AMP-dependent pathways that regulate the expression of the CCAAT enhancer binding proteins beta and delta implicated in adipogenesis. During the pregnancy-lactation period, mother mice were fed either a high-fat diet rich in linoleic acid, a precursor of arachidonic acid (LO diet), or the same isocaloric diet enriched in linoleic acid and alpha-linolenic acid (LO/LL diet). Body weight from weaning onwards, fat mass, epididymal fat pad weight, and adipocyte size at 8 weeks of age were higher with LO diet than with LO/LL diet. In contrast, prostacyclin receptor-deficient mice fed either diet were similar in this respect, indicating that the prostacyclin signaling contributes to adipose tissue development. These results raise the issue of the high content of linoleic acid of i) ingested lipids during pregnancy and lactation, and ii) formula milk and infant foods in relation to the epidemic of childhood obesity.     

Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity

Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.     

Immobilization of native membrane-bound rhodopsin on biosensor surfaces

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.     

The direct effect of leptin on skeletal muscle thermogenesis is mediated by substrate cycling between de novo lipogenesis and lipid oxidation

We report here studies that integrate data of respiration rate from mouse skeletal muscle in response to leptin and pharmacological interference with intermediary metabolism, together with assays for phosphatidylinositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK). Our results suggest that the direct effect of leptin in stimulating thermogenesis in skeletal muscle is mediated by substrate cycling between de novo lipogenesis and lipid oxidation, and that this cycle requires both PI3K and AMPK signaling. This substrate cycling linking glucose and lipid metabolism to thermogenesis provides a novel thermogenic mechanism by which leptin protects skeletal muscle from excessive fat storage and lipotoxicity.