Polyubiquitination of the neurotrophin receptor p75 directs neuronal cell survival

Specific binding of nerve growth factor (NGF) to p75 neurotrophin receptor (p75(NTR)) leads to p75(NTR) polyubiquitination and its subsequent interaction with TRAF6 resulting in neuronal cell survival. However, when the binding of NGF to p75(NTR) was blocked with p75 antiserum, p75(NTR) polyubiquitination and neuronal cell survival were impaired. Results showed that tyrosine phosphorylation of p75(NTR) increased the polyubiquitination of p75(NTR) and contributed to the observed apparent neuroprotective effects. Similar to p75(NTR) polyubiquitination, interaction of TRAF6 with p75(NTR) was NGF/tyrosine phosphorylation dependent suggesting that TRAF6 might function as an E3 ubiquitin ligase. In sum, the results show that specific binding of NGF to p75(NTR) mediates neuronal cell survival.     

Regulation of plasma cholesterol esterification by sphingomyelin: effect of physiological variations of plasma sphingomyelin on lecithin-cholesterol acyltransferase activity

Although sphingomyelin (SM) is the most abundant phospholipid in the plasma, next to phosphatidylcholine (PC), its physiological function in plasma is unclear. Here we employed plasma from various genetic models of mice which naturally differ in their plasma SM/PC ratios, to study the role of SM as a modulator of LCAT, the enzyme responsible for HDL maturation and the synthesis of cholesteryl esters (CE) in normal plasma. Serine palmitoyltransferase deficient mice, and SM synthase deficient mice, both of which have below normal SM/PC ratios, showed significantly elevated LCAT activities when assayed with the endogenous substrates. On the other hand, LDL receptor knockout mice, and apo E knockout mice, both of which have high SM/PC ratios, had markedly reduced (-80%) LCAT activities. The LCAT levels in plasma, as assayed with an exogenous substrate, were similar in all groups, except for a 45% decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 CE all of which are formed by LCAT, and a higher percentage of the atherogenic 18:1 CE which is mainly derived from the action of liver ACAT, showing that in vivo, the contribution of LCAT to plasma CE is reduced while that of liver ACAT is increased. These results show that SM is a physiological modulator of LCAT activity as well as plasma CE composition, and this may contribute to the previously reported pro-atherogenic effect of high plasma SM levels.     

Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.     

Arrangement of the respiratory chain complexes in Saccharomyces cerevisiae supercomplex III2IV2 revealed by single particle cryo-electron microscopy

Here we present for the first time a three-dimensional cryo-EM map of the Saccharomyces cerevisiae respiratory supercomplex composed of dimeric complex III flanked on each side by one monomeric complex IV. A precise fit of the existing atomic x-ray structures of complex III from yeast and complex IV from bovine heart into the cryo-EM map resulted in a pseudo-atomic model of the three-dimensional structure for the supercomplex. The distance between cytochrome c binding sites of complexes III and IV is about 6 nm, which supports proposed channeling of cytochrome c between the individual complexes. The opposing surfaces of complexes III and IV differ considerably from those reported for the bovine heart supercomplex as determined by cryo-EM. A closer association between the individual complex domains at the aqueous membrane interface and larger spaces between the membrane-embedded domains where lipid molecules may reside are also demonstrated. The supercomplex contains about 50 molecules of cardiolipin (CL) with a fatty acid composition identical to that of the inner membrane CL pool, consistent with CL-dependent stabilization of the supercomplex.     

Molecular Interaction Studies of Trimethoxy Flavone with Human Serum Albumin

Background

Human serum albumin (HSA) is the most abundant protein in blood plasma, having high affinity binding sites for several endogenous and exogenous compounds. Trimethoxy flavone (TMF) is a naturally occurring flavone isolated from Andrographis viscosula and used in the treatment of dyspepsia, influenza, malaria, respiratory functions and as an astringent and antidote for poisonous stings of some insects.

Methodology/Principal Findings

The main aim of the experiment was to examine the interaction between TMF and HSA at physiological conditions. Upon addition of TMF to HSA, the fluorescence emission was quenched and the binding constant of TMF with HSA was found to be K_TMF = 1.0±0.01×10³ M⁻¹, which corresponds to −5.4 kcal M⁻¹ of free energy. Micro-TOF Q mass spectrometry results showed a mass increase of from 66,513 Da (free HSA) to 66,823 Da (HAS +Drug), indicating the strong binding of TMF with HSA resulting in decrease of fluorescence. The HSA conformation was altered upon binding of TMF to HSA with decrease in α-helix and an increase in β-sheets and random coils suggesting partial unfolding of protein secondary structure. Molecular docking experiments found that TMF binds strongly with HSA at IIIA domain of hydrophobic pocket with hydrogen bond and hydrophobic interactions. Among which two hydrogen bonds are formed between O (19) of TMF to Arg 410, Tyr 411 and another one from O (7) of TMF to Asn 391, with bond distance of 2.1 Å, 3.6 Å and 2.6 Å, respectively.

Conclusions/Significance

In view of the evidence presented, it is imperative to assign a greater role of HSA''s as a carrier molecule for many drugs to understand the interactions of HSA with TMF will be pivotal in the design of new TMF-inspired drugs.     

Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11^b+ and CD11^b? populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11^b? splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010. © 2010 Wiley‐Liss, Inc.     

BIODEGRADATION OF PHTHALATE ESTERS BY CYANOBACTERIA1

Phthalate esters (PEs) are endocrine‐disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di‐n‐butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos‐aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium‐labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos‐aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first‐order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium‐labeled DBP showed that there were two degradation pathways: C₁₆→ C₁₄→ C₁₂→ C₁₀→ C₈ and C₁₆→ C₁₅→ C₁₃→ C₁₁→ C₉. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested.     

Short-term weight loss in overweight/obese low-income women improves plasma zinc and metabolic syndrome risk factors

Metabolic syndrome is a group of disorders involving obesity, insulin resistance, dyslipidemia and hypertension. Obesity is the most crucial risk factor of metabolic syndrome, because it is known to precede other risk factors. Obesity is also associated with disturbances in the metabolism of the trace mineral, zinc. The overall purpose of this study was to investigate the effects of short-term weight loss on plasma zinc and metabolic syndrome risk factors. An 8-week weight loss intervention study was conducted with 90 low-income overweight/obese mothers, whose youngest child was 1–3 years old. Plasma levels of zinc, glucose, insulin, leptin, triglycerides, total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol were measured and compared at weeks 0 and 8 of the weight loss program. At pre-study, plasma zinc was low in 39% and, within normal values in 46%, of obese/overweight mothers. By the end of intervention, plasma zinc rose by 22% and only 5% of the mothers continued to exhibit low plasma zinc. At post-study, the metabolic syndrome risk factors of waist circumference, HDL cholesterol, and diastolic blood pressure (p < 0.05) showed significant improvements. Plasma zinc increased by a greater margin (67%) in women with low zinc, as compared to those with normal zinc (18%); weight reduction was similar in both the groups. Finally, changes in % body fat were related negatively with changes in plasma zinc (r = ? 0.28, p < 0.05). The circulating levels of zinc, as well as the metabolic syndrome components, showed significant improvements in overweight/obese low-income women after weight loss.