Interference of phenol during quantification of a bacterial lipoprotein

Accurate protein estimation is an essential requirement for any biochemical investigation. The bacterial Braun liporotein (BLP) from E. coli (a Toll-2 receptor ligand) is purified via phenol extraction on the basis of selective extraction of the lipoprotein. The procedure leaves behind the major endotoxin lipopolysaccharide (LPS) that acts through the related Toll-4 receptor. However, as low as 0.00001% of phenol carried over during lipoprotein isolation interferes in the Lowry’s method of protein estimation. A simple gel filtration on sephadex G-50 efficiently separates lipoproteins from phenol thereby avoiding inaccurate protein estimation of the lipoprotein content and making it suitable ligand for Toll-2 receptor.Keywords: Lipoproteins; Lipopolysaccharide (LPS); Lowry's method; Phenol interferenc

Reduced calcification and osteogenic features in advanced atherosclerotic plaques of mice with macrophage-specific loss of TRPC3

Background and aims: Recent in vitro studies have showed that in macrophages, deletion of the non-selective Ca2+-permeable channel TRPC3 impairs expression of the osteogenic protein BMP-2. The pathophysiological relevance of this effect in atherosclerotic plaque calcification remains to be determined. Methods: We used Ldlr−/− mice with macrophage-specific loss of TRPC3 (MacTrpc3−/−/Ldlr−/−) to examine the effect of macrophage Trpc3 on plaque calcification and osteogenic features in advanced atherosclerosis. Results: After 25 weeks on high fat diet, aortic root plaques in MacTrpc3−/−/Ldlr−/− mice showed reduced size, lipid and macrophage content compared to controls. Plaque calcification was decreased in MacTrpc3−/−/Ldlr−/− mice, and this was accompanied by marked reduction in BMP-2, Runx-2 and phospho-SMAD1/5 contents within macrophage-rich areas. Expression of Bmp-2 and Runx-2 was also reduced in bone marrow-derived macrophages from MacTrpc3−/−/Ldlr−/− mice. Conclusions: These findings show that, in advanced atherosclerosis, selective deletion of TRPC3 in macrophages favors plaque regression and impairs the activity of a novel macrophage-associated, BMP-2-dependent mechanism of calcification.Fil: Dube, Prabhatchandra R.. University of Toledo. College of Medicine and Life Sciences; Estados UnidosFil: Chikkamenahalli, Lakshmikanth L.. University of Toledo. College of Medicine and Life Sciences; Estados UnidosFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Vazquez, Guillermo. University of Toledo. College of Medicine and Life Sciences; Estados Unido

Platelet-activating factor and oxidized phosphatidylcholines do not suppress endotoxin-induced pro-inflammatory signaling among human myeloid and endothelial cells

Platelet-activating factor (PAF) and related phospholipid oxidation products termed oxidized phospholipids (OxPLs) promote inflammation. PAF is made in response to bacterial endotoxin-lipopolysaccharide (LPS) that is recognized by Toll-like receptor-4 (TLR-4) whose activation leads to translocation of transcription factor NF-ΚB to the nucleus—a key regulator of multiple pro-inflammatory genes including COX-2 and IL-8. Paradoxically, PAF and OxPLs are claimed to inhibit LPS-mediated signaling, questioning the very pro-inflammatory roles of PAF and OxPLs and anti-inflammatory nature of PAF-acetylhydrolase (PAF-AH), an enzyme that attenuates both PAF and OxPLs signaling. We investigated the effect of PAF and representative OxPLs: 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn- glycero-3-phosphocholine (PGPC) and 1-alkyl-2-butanoyl-sn-glycero-3-phosphocholine PAF (C4 PAF) on LPS-induced expression of NF-ΚB mediated inflammation in isolated human myeloid cells: polymorphonuclear leukocyte (PMNs), monocytes and human umbilical vein endothelial cells (HUVECs). Using intracellular calcium transients, we show that POVPC and PGPC dose-dependently activate the PAF-receptor (PAF-R) in PMNs, that can beblocked by the PAF-R antagonist WEB-2086 and rPAF-AH pre-treatment. All the three cell types express minute or no detectable COX-2 when stimulated with either PAF (0.1 µM) or OxPLs (0.1 µM) alone. While LPS (100 ng/mL) induced expression of COX-2 in all the cell types, pre-activation of PAF-R with PAF (0.1 µM) or OxPLs (0.1 µM) did not suppress LPS (100 ng/mL)-induced COX-2 expression and in fact we obresved incereased PGE2 levels in an NS-398 sensitive manner. In addition, pre-activation of PAF-R significantly augmented LPS (100 ng/mL)-induced IL-8 production in PMNs. Thus, PAF and OXPLs do not suppress the ability of LPS to exert its pro-inflammatory effects in isolated human vascular cells

Ablating astrocyte insulin receptors leads to delayed puberty and hypogonadism in mice.

Insulin resistance and obesity are associated with reduced gonadotropin-releasing hormone (GnRH) release and infertility. Mice that lack insulin receptors (IRs) throughout development in both neuronal and non-neuronal brain cells are known to exhibit subfertility due to hypogonadotropic hypogonadism. However, attempts to recapitulate this phenotype by targeting specific neurons have failed. To determine whether astrocytic insulin sensing plays a role in the regulation of fertility, we generated mice lacking IRs in astrocytes (astrocyte-specific insulin receptor deletion [IRKOGFAP] mice). IRKOGFAP males and females showed a delay in balanopreputial separation or vaginal opening and first estrous, respectively. In adulthood, IRKOGFAP female mice also exhibited longer, irregular estrus cycles, decreased pregnancy rates, and reduced litter sizes. IRKOGFAP mice show normal sexual behavior but hypothalamic-pituitary-gonadotropin (HPG) axis dysregulation, likely explaining their low fecundity. Histological examination of testes and ovaries showed impaired spermatogenesis and ovarian follicle maturation. Finally, reduced prostaglandin E synthase 2 (PGES2) levels were found in astrocytes isolated from these mice, suggesting a mechanism for low GnRH/luteinizing hormone (LH) secretion. These findings demonstrate that insulin sensing by astrocytes is indispensable for the function of the reproductive axis. Additional work is needed to elucidate the role of astrocytes in the maturation of hypothalamic reproductive circuits

Lipopolysaccharide Cross-Tolerance Delays Platelet-Activating Factor-Induced Sudden Death in Swiss Albino Mice: Involvement of Cyclooxygenase in Cross-Tolerance.

Lipopolysaccharide (LPS) signaling through Toll-like receptor-4 (TLR-4) has been implicated in the pathogenesis of many infectious diseases. Some believe that TLR-mediated pathogenicity is due, in part, to the lipid pro-inflammatory mediator platelet-activating factor (PAF), but this has been questioned. To test the direct contribution of PAF in endotoxemia in murine models, we injected PAF intraperitoneally into Swiss albino mice in the presence and absence of LPS. PAF alone (5 μg/mouse) caused death within 15-20 min, but this could be prevented by pretreating mice with PAF-receptor (PAF-R) antagonists or PAF-acetylhydrolase (PAF-AH). A low dose of LPS (5 mg/kg body wt) did not impair PAF-induced death, whereas higher doses (10 or 20 mg/kg body wt) delayed death, probably via LPS cross-tolerance. Cross-tolerance occurred only when PAF was injected simultaneously with LPS or within 30 min of LPS injection. Tolerance does not appear to be due to an abundant soluble mediator. Histologic examination of lungs and liver and measurement of circulating TNF-α and IL-10 levels suggested that the inflammatory response is not diminished during cross-tolerance. Interestingly, aspirin, a non-specific cyclooxygenase (COX) inhibitor, partially blocked PAF-induced sudden death, whereas NS-398, a specific COX-2 inhibitor, completely protected mice from the lethal effects of PAF. Both COX inhibitors (at 20 mg/kg body wt) independently amplified the cross-tolerance exerted by higher dose of LPS, suggesting that COX-derived eicosanoids may be involved in these events. Thus, PAF does not seem to have a protective role in endotoxemia, but its effects are delayed by LPS in a COX-sensitive way. These findings are likely to shed light on basic aspects of the endotoxin cross-tolerance occurring in many disease conditions and may offer new opportunities for clinical intervention

Dose-dependent effect of PAF on mortality in Swiss albino mice.

<p>The indicated amounts of PAF were aliquoted from a stock of 5 mg/mL in methanol, evaporated under a stream of nitrogen, reconstituted in phosphate buffered saline (PBS) containing 0.1% albumin, and sonicated. The animals were divided into 6 groups, each containing 6 animals. Each mouse was intraperitoneally injected with the designated dose of PAF in a total volume of 0.5 mL of PBS containing 0.1% albumin. Control animals received 0.5 mL PBS containing 0.1% albumin. The animals were monitored for survival for up to 6 days. The results are representative of 3 independent trials.</p

LPS cross-tolerance is not transferable.

<p>Mice were divided into 4 groups containing 4 animals each. In the initial phase of this experiment, animals were intraperitoneally injected with 0.5 mL of PBS or with LPS (20 mg/kg) + PAF (5 μg/mouse). These animals were then anesthetized, and their blood was collected 1 h after treatment. In the second phase of this experiment, 100 μL of the serum obtained from the mice treated with PBS or LPS (20 mg/kg body wt) + PAF (5 μg/mouse) was intraperitoneally injected into the other 2 groups of mice 30 min before they were injected with PAF (5 μg/mouse). Half of the mice in the group that originally received PAF (5 μg/mouse) + LPS (20 mg/kg) survived, but the serum from these animals failed to protect the naive animals injected with PAF (5 μg/mouse) 30 min after receiving the serum. The results are representative of 3 individual trials.</p

PAF induces sudden death in Swiss albino mice.

<p>Swiss albino mice were divided into 9 groups containing 6 animals each. The indicated amounts of WEB-2086 or BN-52021 were aliquoted from a stock in DMSO, brought up to a total volume of 0.5 mL in PBS, and injected intraperitoneally into animals 30 min before PAF (5 μg/mouse) was injected. Animals that received rPAF-AH (25 μg/mouse) were intraperitoneally injected with it 30 min before being injected with PAF (5 μg/mouse), as were the animals that received the vehicle for rPAF-AH (sodium citrate, sucrose, pluronic, and Tween-80). All the animals injected with PAF alone (5 μg/mouse) died within 15–20 min. In contrast, all the animals that received either a PAF-R antagonist or rPAF-AH 30 min before being injected with a lethal dose of PAF survived. All animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

Aspirin partially protects Swiss albino mice from PAF-induced lethality and amplifies the cross-tolerance exerted by hyperactivated TLR-4.

<p>Mice were divided into 11 groups. Three groups were intraperitoneally injected with a 10 mg/kg dose of aspirin 30 min before being injected with the indicated doses of PAF, LPS, or both. Three other groups were intraperitoneally injected with a 20 mg/kg dose of aspirin 30 min before receiving PAF, LPS, or both. The 20 mg/kg dose of aspirin partially protected the animals from PAF-induced sudden death, whereas it completely protected the animals from delayed death due to PAF (5 μg/mouse) plus a high dose of LPS (20 mg/kg). The animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p