Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution ...conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
•Are plasma lipids altered differently in cows and goats by milk fat depressing diets?•Cows and goats differ in plasma neutral and polar lipid composition whatever the diet.•Milk fat depressing diets ...increased trans-10 C18:1 in plasma triacylglycerols in cows.•Circulating free and triacylglycerol fatty acids altered by milk fat depressing diets are outlined.•Plasma lipid molecules linked to milk fat depression may help decipher its mechanisms.
This study examines the effects of diets supplemented with various lipids selected to induce divergent milk fat content responses (including a milk fat depression) between dairy cows and goats on plasma lipid composition. The objective was to better understand the mechanisms behind the regulation of milk fat secretion in these two ruminant species. Twelve Holstein cows and 12 Alpine goats were fed a basal diet not supplemented (CTL) or supplemented with corn oil plus wheat starch (COS, 5% DM intake (DMI)), marine algae powder of Schizochytrium sp. (MAP, 1.5% DMI), or hydrogenated palm oil (HPO, 3% DMI), in a replicated 4 × 4 Latin square design, during 28 days. On day 27, blood samples were collected for lipid analysis. Plasma lipid classes were quantified by high-performance thin-layer chromatography, with triacylglycerol (TAG) and free fatty acid (FFA) fractions analysed for FA composition by GLC. Plasma molecular species of TAG and ceramides were determined by HPLC–high-resolution MS and by liquid chromatography–triple quadrupole, respectively. Irrespective of diet, plasma total lipid content was higher in cows than goats (+61%), and TAG concentration was higher in goats than cows (+157%). In cows, conversely to goats, COS increased the trans-10 C18:1 proportion in the free FA (+248%) and the TAG (+195%) fractions. In cows and goats, MAP induced increases in cholesterol esters, cholesterol and phospholipids compared to CTL and changes in the plasma free FA and FA of TAG profiles. In both ruminant species, the concentrations of the lipid fractions were unchanged by HPO compared to CTL. Our results point to species specificities and different diet effects in plasma concentrations and compositions of lipid fractions in cows and goats. These new data highlight how diets, that induce large variations in milk fat secretions, affect the plasma lipid classes available for milk fat synthesis.
Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell ...death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Essentials
The role of ATP‐binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized.
We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and ...two Tangier patients.
ABCA1‐deficient platelets exhibit reduced positive feedback loop mechanisms.
This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1.
Summary
Background
The ATP‐binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A‐1 to high‐density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized.
Objective
To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition.
Methods and results
We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1‐deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium‐induced phosphatidylserine exposure. Interestingly, ABCA1‐deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients.
Conclusions
Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1‐independent mechanism.
We have recently reported that cytostatic concentrations of the microsomal antiestrogen-binding site (AEBS) ligands, such as PBPE (N-pyrrolidino-(phenylmethyphenoxy)-ethanamine,HCl) and tamoxifen, ...induced differentiation characteristics in breast cancer cells through the accumulation of post-lanosterol intermediates of cholesterol biosynthesis. We show here that exposure of MCF-7 (human breast adenocarcinoma cell line) cells to higher concentrations of AEBS ligands triggered active cell death and macroautophagy. Apoptosis was characterized by Annexin V binding, chromatin condensation, DNA laddering and disruption of the mitochondrial functions. We determined that cell death was sterol- and reactive oxygen species-dependent and was prevented by the antioxidant vitamin E. Macroautophagy was characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin-1 and the stimulation of autophagic flux. We established that macroautophagy was sterol- and Beclin-1-dependent and was associated with cell survival rather than with cytotoxicity, as blockage of macroautophagy sensitized cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and shows a mechanism that explains the induction of autophagy in MCF-7 cells by tamoxifen and other selective estrogen receptor modulators.
Purpose
To evaluate the lipidomic study impact on AMD diagnosis, screening, etiopathogenie and interrelations,correlations between those 2 entities.
Methods
AMD: 30 AMD patients. 3 Groups: A:10 first ...stage AMD patients,B:10 Atrophy AMD patients, C:10 Neovascular AMD patients. Ophthalmologic exam:ETDRS visual acuity (VA), complete ophthalmic examination,Fundus examination, autofluorescence imaging (FAF), optical coherence tomography (Spectral Domain OCT) and fluorescein angiography (FA) and ICG when Neovascular complication.Lipidomic Study: Blood tests and analysis, all lipids qualitative, quantitative analysis, all the same for all patients, whatever group. Blood test is done during ophthalmologic exam. Plasma congelation “snap frost” in liquid nitrogen after total blood centrifugation, then liquid‐liquid extraction for lipids analysis:neutral lipid by GC, as well as fatty acid but after BF3 methanol derivation,phospholipids by LC‐MS directly,as sphingolipids but firstly hydrolysed. Polyinsatured fatty acids metabolites preparation: protein precipitation then pre‐concentration by SPE(solid phase extraction) before m'analyses by LC‐MS.
Results
Analysis will determine qualitative,quantitative lipids values in each patients group, each of them proportion, characterization, singularity; so, characterization, prevalence, specifics of and for each group. Lipidomic study’ evaluation, identification, classification in AMD patients groups allow AMD screening,follow‐up, particularly according to AMD type and stage. Lipidomic study have biomarker feature, let AMD prevention, etiopathogenic concept.
Conclusions
Lipidomics study and so better AMD characterization allow better diagnosis, follow‐up, screening of AMD. Interrelations and correlations between AMD and Lipidomics lead to better etiopathogenie understanding and therapeutics prospects.
Purpose
To evaluate the lipidomic study impact on AMD diagnosis, screening, etiopathogenie and interrelations, correlations between those 2 entities
Methods
AMD:30 AMD patients. 3 Groups: A:10 first ...stage AMD patients, B:10 Atrophy AMD patients, C:10 Neovascular AMD patients. Ophthalmologic exam:ETDRS visual acuity (VA), complete ophthalmic examination, Fundus examination, autofluorescence imaging (FAF), optical coherence tomography (Spectral Domain OCT) and fluorescein angiography (FA) and ICG when Neovascular complication. Lipidomic Study:Blood tests and analysis, all lipids qualitative, quantitative analysis, all the same for all patients, whatever group. Blood test is done during ophthalmologic exam. Plasma congelation “snap frost” in liquid nitrogen after total blood centrifugation, then liquid‐liquid extraction for lipids analysis:neutral lipid by GC, as well as fatty acid but after BF3 methanol derivation, phospholipids by LC‐MS directly, as sphingolipids but firstly hydrolysed. Polyinsatured fatty acids metabolites preparation: protein precipitation then pre‐concentration by SPE (solid phase extraction) before m'analyses by LC‐MS
Results
Analysis will determine qualitative, quantitative lipids values in each patients group, each of them proportion, characterization, singularity; so, characterization, prevalence, specifics of and for each group. Lipidomic study'evaluation, identification, classification in AMD patients groups allow AMD screening, follow‐up, particularly according to AMD type and stage. Lipidomic study have biomarker feature, let AMD prevention, etiopathogenic concept.
Conclusions
Lipidomics study and so better AMD characterization allow better diagnosis, follow‐up, screening of AMD. Interrelations and correlations between AMD and Lipidomics lead to better etiopathogenie understanding and therapeutics prospects.
Introduction Le vieillissement se caractérise par une perte de masse musculaire et un gain de masse grasse notamment viscérale. L’augmentation de la résistance à l’insuline due à l’obésité viscérale ...peut s’expliquer par deux phénomènes, la lipotoxicité et l’inflammation. L’objectif était de mesurer l’effet de l’obésité au cours du vieillissement sur la sensibilité à l’insuline, l’inflammation et la lipotoxicité et les conséquences sur la synthèse protéique musculaire. Matériels et méthodes Durant 10 semaines, 34 rats WISTAR âgés de 6 ou 25 mois ont reçu un régime normo- (JNL, VNL) ou hyper-lipidique/hyper-calorique (JHL, VHL). Un test de tolérance au glucose a été réalisé afin d’évaluer la sensibilité à l’insuline. Le profil inflammatoire a été évalué en mesurant la concentration plasmatique du récepteur soluble du TNF-α (sTNF-R2). Les taux de céramides, de diacylglycerols et de triglycérides ont été dosés au sein des muscles. La synthèse protéique musculaire (FSR) a été calculée à partir de l’enrichissement en 13C valine des protéines. Résultats Le groupe VHL était le plus insulino-résistant (p < 0,05 vs. JNL, JHL et VNL). La concentration plasmatique en sTNF-R2 pour le groupe VHL était supérieure à celle des JNL, JHL, et VNL (p < 0,05). La concentration des dérivés lipidiques musculaires était augmentée dans le groupe VHL comparativement aux trois autres groupes (p < 0,05). Le FSR des protéines musculaires était diminué de 18,6% dans le groupe VHL (p < 0,05) comparativement au groupe VNL. Conclusion Nous observons une accumulation liée à l’âge des dérivés lipidiques au sein des muscles des rats; ce phénomène étant aggravé par le régime HL. L’augmentation de l’inflammation chez les VHL pourrait être responsable de la dérégulation de la fonction tampon du tissu adipeux entraînant un stockage des lipides au sein du muscle. Les conséquences seraient une augmentation de l’insulinorésistance et une diminution de la synthèse protéique musculaire.
Background & Aims In mice, activation of the transient receptor potential cation channels (TRP) TRPV1, TRPV4, and TRPA1 causes visceral hypersensitivity. These receptors and their agonists might be ...involved in development of irritable bowel syndrome (IBS). We investigated whether polyunsaturated fatty acid (PUFA) metabolites, which activate TRPs, are present in colon tissues from patients with IBS and act as endogenous agonists to induce hypersensitivity. Methods We analyzed colon biopsy samples from 40 patients with IBS (IBS biopsies) and 11 healthy individuals undergoing colorectal cancer screening (controls), collected during colonoscopy at the University of Bologna, Italy. Levels of the PUFA metabolites that activate TRPV1 (12-hydroperoxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 5-hydroxyeicosatetraenoic acid, and leukotriene B4), TRPV4 (5,6-epoxyeicosatrienoic acid EET and 8,9-EET), and TRPA1 (PGA1 , 8-iso-prostaglandin A2 , and 15-deoxy-Δ-prostaglandin J2 ) were measured in biopsies and their supernatants using liquid chromatography and tandem mass spectrometry; we also measured levels of the PUFA metabolites prostaglandin E2 (PGE2 ) and resolvins. C57Bl6 mice were given intrathecal injections of small interfering RNAs to reduce levels of TRPV4, or control small interfering RNAs, along with colonic injections of biopsy supernatants; visceral hypersensitivity was measured based on response to colorectal distension. Mouse sensory neurons were cultured and incubated with biopsy supernatants and lipids extracted from biopsies or colons of mice. Immunohistochemistry was used to detect TRPV4 in human dorsal root ganglia samples (from the National Disease Research Interchange). Results Levels of the TRPV4 agonist 5,6-EET, but not levels of TRPV1 or TRPA1 agonists, were increased in IBS biopsies compared with controls; increases correlated with pain and bloating scores. Supernatants from IBS biopsies, but not from controls, induced visceral hypersensitivity in mice. Small interfering RNA knockdown of TRPV4 in mouse primary afferent neurons inhibited the hypersensitivity caused by supernatants from IBS biopsies. Levels of 5,6-EET and 15-HETE were increased in colons of mice with, but not without, visceral hypersensitivity. PUFA metabolites extracted from IBS biopsies or colons of mice with visceral hypersensitivity activated mouse sensory neurons in vitro, by activating TRPV4. Mouse sensory neurons exposed to supernatants from IBS biopsies produced 5,6-EET via a mechanism that involved the proteinase-activated receptor-2 and cytochrome epoxygenase. In human dorsal root ganglia, TPV4 was expressed by 35% of neurons. Conclusions Colon tissues from patients with IBS have increased levels of specific PUFA metabolites. These stimulate sensory neurons from mice and generate visceral hypersensitivity via activation of TRPV4.
Background and Purpose
Long‐term intake of dietary fatty acids is known to predispose to chronic inflammation, but their effects on acute intestinal ischaemia/reperfusion (I/R) injury is unknown. The ...aim of this study was to determine the consequences of a diet rich in n‐3 or n‐6 polyunsaturated fatty acids (PUFA) on intestinal I/R‐induced damage.
Experimental Approach
Mice were fed three different isocaloric diets: a balanced diet used as a control and two different PUFA‐enriched diets, providing either high levels of n‐3 or of n‐6 PUFA. Intestinal injury was evaluated after intestinal I/R. PUFA metabolites were quantitated in intestinal tissues by LC‐MS/MS.
Key Results
In control diet‐fed mice, intestinal I/R caused inflammation and increased COX and lipoxygenase‐derived metabolites compared with sham‐operated animals. Lipoxin A4 (LxA4) was significantly and selectively increased after ischaemia. Animals fed a high n‐3 diet did not display a different inflammatory profile following intestinal I/R compared with control diet‐fed animals. In contrast, intestinal inflammation was decreased in the I/R group fed with high n‐6 diet and level of LxA4 was increased post‐ischaemia compared with control diet‐fed mice. Blockade of the LxA4 receptor (Fpr2), prevented the anti‐inflammatory effects associated with the n‐6 rich diet.
Conclusions and Implications
This study indicates that high levels of dietary n‐6, but not n‐3, PUFAs provides significant protection against intestinal I/R‐induced damage and demonstrates that the endogenous production of LxA4 can be influenced by diet.
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