Functional platelet-derived mitochondria induce the release of human neutrophil microvesicles.

Inflammation is an essential process of host defense against infections, illness, or tissue damage. Polymorphonuclear neutrophils (PMN) are among the first immune cells involved in acute inflammatory responses and are on the front line in the fight against bacterial infections. In the presence of bacterial fragments, PMN release inflammatory mediators, enzymes, and microvesicles in the extracellular milieu to recruit additional immune cells required to eliminate the pathogens. Recent evidence shows that platelets (PLTs), initially described for their role in coagulation, are involved in inflammatory responses. Furthermore, upon activation, PLT also release functional mitochondria (freeMitos) within their extracellular milieu. Mitochondria share characteristics with bacterial and mitochondrial damage-associated molecular patterns, which are important contributors in sterile inflammation processes. Deep sequencing transcriptome analysis demonstrates that freeMitos increase the mitochondrial gene expression in PMN. However, freeMitos do not affect the mitochondrial-dependent increase in oxygen consumption in PMN. Interestingly, freeMitos significantly induce the release of PMN-derived microvesicles. This study provides new insight into the role of freeMitos in the context of sterile inflammation.

Dietary omega-3 fatty acids modulate the production of platelet-derived microvesicles in an in vivo inflammatory arthritis model.

PURPOSE: The aim of this study was to investigate the effects of different ω-3 polyunsaturated fatty acid (PUFA) enriched diets, including a novel renewable plant source of ω-3 fatty acids (Buglossoides arvensis), on the development and progression of rheumatoid arthritis (RA). METHODS: RA was induced in mice consuming experimental diets using the K/BxN model. The experimental diets consisted of either a western control diet (control), diets containing B. arvensis oil or fish oil. The effects of the diets on platelets, platelet microvesicles (PMVs), and inflammatory markers such as clinical index, ankle thickness and cytokine/chemokine release were measured. RESULTS: While ω-3 PUFA-enriched diets did not prevent the development of arthritis in the K/BxN model, a significant decrease in ankle swelling was observed compared to the control group. Platelets isolated from mice consuming either low content of B. arvensis oil or fish oil diets exhibited significantly decreased PMVs production compared to mice consuming the control diet. CONCLUSION: Our study provides insight into the contribution of ω-3 PUFA supplementation in modulating the pro-inflammatory phenotype of platelets in RA pathology. Furthermore, our study suggests that low concentrations of dietary B. arvensis oil may have similar anti-inflammatory potential seen with dietary fish oil supplementation.

Algerian Propolis from Distinct Geographical Locations: Chemical Profiles, Antioxidant Capacity, Cytotoxicity and Inhibition of 5-Lipoxygenase Product Biosynthesis.

Propolis was collected from honeybee hives in three geographically distinct Algerian climates and extracts were characterized for composition and bioactivity. Bees were identified as native subspecies using an in-silico DraI mtDNA COI-COII test. Over 20 compounds were identified in extracts by LC-MS. Extracts from the Medea region were more enriched in phenolic content (302±28 mg GAE/g of dry extract) than those from Annaba and Ghardaia regions. Annaba extracts had the highest flavonoid content (1870±385 mg QCE/g of dry extract). Medea extracts presented the highest free-radical scavenging activity (IC50=13.5 µg/mL) using the DPPH radical assay while Ghardaia extracts from the desert region were weak (IC50>100 µg/mL). Antioxidant activities measured using AAPH oxidation of linoleic acid were similar in all extracts with IC50 values ranging from 2.9 to 4.9 µg/mL. All extracts were cytotoxic (MTT assay) and proapoptotic (Annexin-V) against human leukemia cell lines in the low µg/mL range, although the Annaba extract was less active against the Reh cell line. Extracts inhibited cellular 5-lipoxygenase product biosynthesis with IC50 values ranging from 0.6 to 3.2 µg/mL. Overall, examined propolis extracts exhibited significant biological activity that warrant further characterization in cellular and in vivo models.

Single and multiple inhibitors of the biosynthesis of 5-, 12-, 15-lipoxygenase products derived from cinnamyl-3,4-dihydroxy-α-cyanocinnamate: Synthesis and structure-activity relationship.

The involvement of lipoxygenases in various pathologies, combined with the unavailability of safe and effective inhibitors of the biosynthesis of their products, is a source of inspiration for the development of new inhibitors. Based on a structural analysis of known inhibitors of lipoxygenase products biosynthesis, a comprehensive structure-activity study was carried out, which led to the discovery of several novel compounds (16a-c, 17a) demonstrating promising potency to inhibit the biosynthesis of products of 5-, 12- and 15-LO. Compounds 16b and 16c outperformed zileuton (1), the only FDA-approved 5-LO inhibitor, as well as known inhibitors such as caffeic acid phenethyl ester (CAPE (2)) and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC (4)). However, the introduction of a cyano group at the α-position of the carbonyl abolished the activity. Compounds 16a and 17a also inhibited the biosynthesis of 12- and 15-LO products. Compounds 16a, 17a far surpassed baicalein, a known 12-LO inhibitor, as inhibitors of 12-LO products biosynthesis. Compound 17a and CDC (4) showed equivalent inhibition of LO products, proposing that the double bond in the ester moiety is not necessary for the inhibitory activity. The introduction of the cyano group, as in compound 17a, at the α-position of the carbonyl in compound 16a significantly reduced the inhibitory activity against the biosynthesis of 15-LO products. In addition to the interactions with residues His372 and Phe421 also found with zileuton and CAPE, compounds 16a and 16c each interact with residue His367 as shown by molecular docking. This new interaction may explain their high affinity with the 5-LO active site.

Loss of detection of fatty acid-metabolizing proteins in Western blot analyses - Impact of sample heating.

When performing western blots for protein detection using the classical Laemmli method, experimenters often encounter difficulties with the detection of transmembrane proteins involved in lipid or fatty acid metabolism. A crucial phase in sample preparation is heating the samples to 100 °C in a Laemmli sample buffer containing SDS before separation by polyacrylamide gel electrophoresis (PAGE). In the current study, the analysis of several proteins was performed following modifications of the heating step during sample preparation. Multiple samples of the human Jurkat cell line were prepared using commercial or homemade Laemmli sample buffer. Samples were subjected to incubation at different temperatures for varying periods of time prior to separation by SDS-PAGE, transfer onto PVDF membranes and detection with specific antibodies. In samples incubated at temperatures of 25 °C, 40 °C, 70 °C and 100 °C, detection of the transmembrane protein elongase of long chain fatty acids 5 (ELOVL5) significantly decreased with temperature to a near total absence of signal at 100 °C. Heating (100 °C) the samples even for 1 min resulted in significant loss of ELOVL5 band intensity that was associated with the appearance of higher molecular weight immunoreactive materials. Loss of ELOVL5 band intensity was also observed with heating of samples at 100 °C prepared from HepG2, HEK293, MCF-7 and SKRB cells. The robust induction of ELOVL5 in stimulated primary T cells was not detected when sample were heated. The detection of fatty acid-metabolizing enzymes stearoyl-CoA desaturase-1 and long-chain-fatty-acid-CoA ligases 3 and 4 showed bands with significantly less intensity after heating at 100 °C compared to samples prepared at room temperature. Heating samples at 100 °C did not affect the detection of transmembrane proteins ERBB2 and five-lipoxygenase activating protein, or the soluble 5-lipoxygenase protein. Overall, the number of transmembrane passes of a protein was not predictive of loss of band intensity after heating, however this study indicates that sample heating can drastically affect the ability to detect proteins following separation by SDS-PAGE. This has implications for any detection methods that follow SDS-PAGE.

Structure-Activity Relationship Studies of New Sinapic Acid Phenethyl Ester Analogues Targeting the Biosynthesis of 5-Lipoxygenase Products: The Role of Phenolic Moiety, Ester Function, and Bioisosterism.

Sinapic acid is found in many edible plants and fruits, such as rapeseed, where it is the predominant phenolic compound. New sinapic acid phenethyl ester (SAPE) analogues were synthesized and screened as inhibitors of the biosynthesis of 5-lipoxygenase (5-LO) in stimulated HEK293 cells and polymorphonuclear leukocytes (PMNL). Inhibition of leukotriene biosynthesis catalyzed by 5-LO is a validated therapeutic strategy against certain inflammatory diseases and allergies. Unfortunately, the only inhibitor approved to date has limited clinical use because of its poor pharmacokinetic profile and liver toxicity. With the new analogues synthesized in this study, the role of the phenolic moiety, ester function, and bioisosterism was investigated. Several of the 34 compounds inhibited the biosynthesis of 5-LO products, and 20 compounds were 2-11 times more potent than zileuton in PMNL, which are important producers of 5-LO products. Compounds 5i (IC50: 0.20 µM), 5l (IC50: 0.20 µM), and 5o (IC50: 0.21 µM) bearing 4-trifluoromethyl, methyl, or methoxy substituent at meta-position of the phenethyl moiety were 1.5 and 11.5 times more potent than SAPE (IC50: 0.30 µM) and zileuton (IC50: 2.31 µM), respectively. Additionally, compound 9 (IC50: 0.27 µM), which was obtained after acetylation of the 4-hydroxyl of SAPE, was equivalent to SAPE and 8 times more active than zileuton. Furthermore, compound 20b (IC50: 0.27 µM) obtained after the bioisosteric replacement of the ester function of SAPE by the 1,2,4-oxadiazole heterocycle was equivalent to SAPE and 8 times more active than zileuton. Thus, this study provides a basis for the rational design of new molecules that could be developed further as anti 5-LO therapeutics.

New Zileuton-Hydroxycinnamic Acid Hybrids: Synthesis and Structure-Activity Relationship towards 5-Lipoxygenase Inhibition.

A novel series of zileuton-hydroxycinnamic acid hybrids were synthesized and screened as 5-lipoxygenase (5-LO) inhibitors in stimulated HEK293 cells and polymorphonuclear leukocytes (PMNL). Zileuton's (1) benzo[b]thiophene and hydroxyurea subunits combined with hydroxycinnamic acid esters' ester linkage and phenolic acid moieties were investigated. Compound 28, bearing zileuton's (1) benzo[b]thiophene and sinapic acid phenethyl ester's (2) α,ß-unsaturated phenolic acid moiety 28, was shown to be equipotent to zileuton (1), the only clinically approved 5-LO inhibitor, in stimulated HEK293 cells. Compound 28 was three times as active as zileuton (1) for the inhibition of 5-LO in PMNL. Compound 37, bearing the same sinapic acid (3,5-dimethoxy-4-hydroxy substitution) moiety as 28, combined with zileuton's (1) hydroxyurea subunit was inactive. This result shows that the zileuton's (1) benzo[b]thiophene moiety is essential for the inhibition of 5-LO product biosynthesis with our hydrids. Unlike zileuton (1), Compound 28 formed two π-π interactions with Phe177 and Phe421 as predicted when docked into 5-LO. Compound 28 was the only docked ligand that showed a π-π interaction with Phe177 which may play a part in product specificity as reported.

Extraction, Antioxidant Capacity, 5-Lipoxygenase Inhibition, and Phytochemical Composition of Propolis from Eastern Canada.

Soxhlet (SE), microwave-assisted (MAE) and ultrasound-assisted (UAE) extraction were compared using ten extraction solvents for their efficiency to extract phenolic and flavonoid antioxidants from Eastern Canada propolis. Extracts were compared for total phenolic (TPC) and total flavonoid (TFC) content, and radical scavenging activities. Anti-inflammatory activity through inhibition of 5-lipoxygenase (5-LO) products biosynthesis in HEK293 cells was also evaluated. The results showed that SE extracts using polar solvents had the highest TPC and TFC. Extracts obtained with ethanol, methanol and acetone were effective free radical scavengers, and showed 5-LO inhibition similar to zileuton. UAE was an effective extraction method since the extracts obtained were comparable to those using SE and the MAE while being done at room temperature. With UAE, extracts of less polar solvents showed similar free radical scavenging and 5-LO inhibition to extracts of much more polar solvents such as methanol or ethanol. Reversed-phase liquid chromatography tandem mass spectrometry confirmed the presence of 21 natural compounds in the propolis extracts based on the comparison of intact mass, chromatographic retention time and fragmentation patterns derived from commercial analytical standards. The current study is the first of its kind to concurrently investigate solvent polarity as well as extraction techniques of propolis.

Identification of Peracetylated Quercetin as a Selective 12-Lipoxygenase Pathway Inhibitor in Human Platelets.

The inflammatory response is necessary for the host's defense against pathogens; however, uncontrolled or unregulated production of eicosanoids has been associated with several types of chronic inflammatory diseases. Thus, it is not surprising that enzymes implicated in the production of eicosanoids have been strategically targeted for potential therapeutic approaches. The 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] lipid mediator is among inflammatory molecules that are abundantly produced in various diseases and is primarily biosynthesized via the 12(S)-lipoxygenase pathway. The effects of the abundance of 12(S)-HETE and its contribution to several chronic inflammatory diseases have been well studied over the last few years. While most developed compounds primarily target the 5-lipoxygenase (5-LO) or the cyclooxygenase (COX) pathways, very few compounds selectively inhibiting the 12-lipoxygenase (12-LO) pathway are known. In this study, we examined whether the distribution of hydroxyl groups among flavones could influence their potency as 12-LO inhibitors. Using human platelets, the human embryonic kidney 293 (HEK293) cell line expressing 5-LO, and human polymorphonuclear leukocytes (PMNLs) we investigated the effects of these compounds on several inflammatory pathways, namely, 12-LO, 5-LO, and COX. Using high-resolution respirometry and flow cytometry, we also evaluated some normal cell functions that could be modulated by our compounds. We identified a peracetylated quercetin (compound 6) that exerts potent inhibitory activity toward the platelet 12-LO pathway (IC50 = 1.53 µM) while having a lesser affinity toward the COX pathway. This study characterizes the peracetylated quercetin (compound 6) as a more selective platelet-type 12-LO inhibitor than baicalein, with no measurable nontargeted effects on the platelet's activation or overall cell's oxygen consumption.

Impact of maternal iron deficiency on the auditory functions in the young and adult guinea pig.

OBJECTIVES: The aim of this study was to evaluate the hearing function in the guinea pig offspring at post-natal day (PNd) 24 and PNd84 born from dams suffering from iron deficiency during pregnancy and lactation by using the auditory brainstem response (ABR). METHOD: Female guinea pigs (n = 24 per dietary group) were fed an iron sufficient (IS) diet (114 mg/kg) or an iron deficient (ID) diet (11.7 mg/kg) during the gestation and lactation periods. Pups in both groups were weaned at PNd9 and given the IS diet. The hematocrit level was measured at every trimester of pregnancy and at the day of sacrifice in dams and at PNd24 and PNd84 in pups. The animal body weight was measured on every second day until the day of sacrifice. The ABR was used in pups to measure the hearing threshold using a broad range of stimulus intensities and latency at 100 and 80 dB in response to 2, 4, 8, 16, and 32 kHz tone pips at PNd24 and 84. RESULTS AND DISCUSSION: No significant difference between dietary groups was measured in hearing threshold and absolute latencies in pups at PNd24 and PNd84. Although the ID offspring (n = 16) did not differ in brainstem transmission times (BTTs) at 80 dB compare to the IS siblings (n = 25) at PNd24, they showed significant delayed inter-peak latency (IPL) I-IV at 100 dB suggesting a delayed BTT. At PNd84, the latency of all peaks including IPL I-IV at 80 and 100 dB significantly decreased and was also similar in pups from both dietary groups suggesting a better brain maturation. This is the first study investigating the long-term impact of maternal iron deficiency on the auditory functions in the guinea pig offspring during early development to adulthood.

Polyunsaturated fatty acid elongation and desaturation in activated human T-cells: ELOVL5 is the key elongase.

PUFAs are important constituents of membrane glycerophospholipids. However, changes in the capacities to incorporate and metabolize PUFAs when cells enter the cell cycle have not been thoroughly studied. In this study, differences in the incorporation and metabolism of exogenous PUFAs in resting and proliferating primary human T-cells and in the Jurkat cell line were measured. Overall, proliferating T-cells and Jurkat cells had a greater capacity to incorporate and elongate exogenous 18- and 20-carbon PUFAs compared with resting T-cells. Proliferating T-cells and Jurkat cells also showed a greater capacity to desaturate 18-carbon PUFA substrates. Consistent with these observations, a significant increase in the expression of fatty acid desaturase (FADS) 1, FADS2, and elongation of very long chain fatty acids protein (ELOVL) 5 was measured in proliferating T-cells compared with resting T-cells. No quantifiable ELOVL2 was measured. Knockdown of ELOVL5 in T-cells and Jurkat cells significantly affected cellular monounsaturated and PUFA profiles and strongly impaired the elongation of 18- and 20-carbon PUFAs. In conclusion, the induction of proliferation in human T-cells is associated with a significant increase in the capacity to take up and metabolize exogenous PUFAs, and ELOVL5 is responsible for the elongation of 18- and 20-carbon PUFAs in these cells.

Ground flaxseed reverses protection of a reduced-fat diet against Citrobacter rodentium-induced colitis.

Flaxseed is high in ω-3 polyunsaturated fatty acids, fiber, and lignans known to lower cholesterol levels. However, its use for prevention or treatment of inflammatory bowel diseases has yielded mixed results, perhaps related to dietary interactions. In this study, we evaluated the impact of ground flaxseed supplementation on the severity of Citrobacter rodentium-induced colitis in the setting of either a high-fat (HF, ~36%kcal) or reduced-fat (RF, ~12%kcal) diet. After weaning, C57BL/6 mice ( n = 8-15/treatment) were fed ground flaxseed (7 g/100 g diet) with either HF (HF Flx) or RF (RF Flx) diets for 4 wk before infection with C. rodentium or sham gavage. Weight changes, mucosal inflammation, pathogen burden, gut microbiota composition, tissue polyunsaturated fatty acids, and cecal short-chain fatty acids were compared over a 14-day infection period. The RF diet protected against C. rodentium-induced colitis, whereas the RF Flx diet increased pathogen burden, exacerbated gut inflammation, and promoted gut dysbiosis. When compared with the RF diet, both HF and HF Flx diets resulted in more severe pathology in response to C. rodentium infection. Our findings demonstrate that although an RF diet protected against C. rodentium-induced colitis and associated gut dysbiosis in mice, beneficial effects were diminished with ground flaxseed supplementation. NEW & NOTEWORTHY Our results demonstrate a strong protective effect of a reduced-fat diet against intestinal inflammation, dysbiosis, and pathogen burden during Citrobacter rodentium-induced colitis. However, ground flaxseed supplementation in the setting of a reduced-fat diet exacerbated colitis despite higher levels of intestinal n-3 polyunsaturated fatty acids and cecal short-chain fatty acids.

17ß-estradiol-induced ACSL4 protein expression promotes an invasive phenotype in estrogen receptor positive mammary carcinoma cells.

Long chain acyl-CoA synthase-4 (ACSL4) expression has been associated with an aggressive phenotype in breast carcinoma cells, whereas its role in ERα-positive breast cancer has not been studied. ACSL4 prefers 20-carbon polyunsaturated fatty acid (PUFA) substrates, and along with other ACSLs has been associated with cellular uptake of exogenous fatty acids. 17ß-estradiol induces proliferation and invasive capacities in ERα+ve breast carcinoma that is associated with modifications of cellular lipid metabolism. In this study, treatment of steroid-starved ERα-positive MCF-7 and T47D mammary carcinoma cells with 17ß-estradiol resulted in increased cellular uptake of the PUFA arachidonic acid (AA) and eicosapentaenoic acid (EPA), important building blocks for cellular membranes, and increased ACSL4 protein levels. There was no change in the expression of the ACSL1, ACSL3 and ACSL6 protein isotypes. Increased ACSL4 protein expression was not accompanied by changes in ACSL4 mRNA expression, but was associated with a significant increase in the protein half-life compared to untreated cells. ERα silencing reversed the impact of 17ß-estradiol on ACSL4 protein levels and half-life. Silencing of ACSL4 eliminated the 17ß-estradiol-induced increase in AA and EPA uptake, as well as the 17ß-estradiol-induced cell migration, proliferation and invasion capacities. ASCL4 silencing also prevented the 17ß-estradiol induced increases in p-Akt and p-GSK3ß, and decrease in E-cadherin expression, important events in epithelial to mesenchymal transition. Taken together, these results demonstrate that ACSL4 is a target of 17ß-estradiol-stimulated ERα and is required for the cellular uptake of exogenous PUFA and the manifestation of a more malignant phenotype in ERα+ve breast carcinoma cells.

New Hydroxycinnamic Acid Esters as Novel 5-Lipoxygenase Inhibitors That Affect Leukotriene Biosynthesis.

Leukotrienes are inflammatory mediators that actively participate in the inflammatory response and host defense against pathogens. However, leukotrienes also participate in chronic inflammatory diseases. 5-lipoxygenase is a key enzyme in the biosynthesis of leukotrienes and is thus a validated therapeutic target. As of today, zileuton remains the only clinically approved 5-lipoxygenase inhibitor; however, its use has been limited due to severe side effects in some patients. Hence, the search for a better 5-lipoxygenase inhibitor continues. In this study, we investigated structural analogues of caffeic acid phenethyl ester, a naturally-occurring 5-lipoxygenase inhibitor, in an attempt to enhance the inhibitory activity against 5-lipoxygenase and determine structure-activity relationships. These compounds were investigated for their ability to attenuate the biosynthesis of leukotrienes. Compounds 13 and 19, phenpropyl and diphenylethyl esters, exhibited significantly enhanced inhibitory activity when compared to the reference molecules caffeic acid phenethyl ester and zileuton.

Substituted Caffeic and Ferulic Acid Phenethyl Esters: Synthesis, Leukotrienes Biosynthesis Inhibition, and Cytotoxic Activity.

Glioblastoma multiforme (GBM) is an aggressive brain tumor that correlates with short patient survival and for which therapeutic options are limited. Polyphenolic compounds, including caffeic acid phenethyl ester (CAPE, 1a), have been investigated for their anticancer properties in several types of cancer. To further explore these properties in brain cancer cells, a series of caffeic and ferulic acid esters bearing additional oxygens moieties (OH or OCH3) were designed and synthesized. (CAPE, 1a), but not ferulic acid phenethyl ester (FAPE, 1b), displayed substantial cytotoxicity against two glioma cell lines. Some but not all selected compounds derived from both (CAPE, 1a) and (FAPE, 1b) also displayed cytotoxicity. All CAPE-derived compounds were able to significantly inhibit 5-lipoxygenase (5-LO), however FAPE-derived compounds were largely ineffective 5-LO inhibitors. Molecular docking revealed new hydrogen bonds and π-π interactions between the enzyme and some of the investigated compounds. Overall, this work highlights the relevance of exploring polyphenolic compounds in cancer models and provides additional leads in the development of novel therapeutic strategies in gliomas.

On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid.

Alkyne and azide analogs of natural compounds that can be coupled to sensitive tags by click chemistry are powerful tools to study biological processes. Arachidonic acid (AA) is a FA precursor to biologically active compounds. 19-Alkyne-AA (AA-alk) is a sensitive clickable AA analog; however, its use as a surrogate to study AA metabolism requires further evaluation. In this study, AA-alk metabolism was compared with that of AA in human cells. Jurkat cell uptake of AA was 2-fold greater than that of AA-alk, but significantly more AA-Alk was elongated to 22:4. AA and AA-alk incorporation into and remodeling between phospholipid (PL) classes was identical indicating equivalent CoA-independent AA-PL remodeling. Platelets stimulated in the pre-sence of AA-alk synthesized significantly less 12-lipoxygenase (12-LOX) and cyclooxygenase products than in the presence of AA. Ionophore-stimulated neutrophils produced significantly more 5-LOX products in the presence of AA-alk than AA. Neutrophils stimulated with only exogenous AA-alk produced significantly less 5-LOX products compared with AA, and leukotriene B4 (LTB4)-alk was 12-fold less potent at stimulating neutrophil migration than LTB4, collectively indicative of weaker leukotriene B4 receptor 1 agonist activity of LTB4-alk. Overall, these results suggest that the use of AA-alk as a surrogate for the study of AA metabolism should be carried out with caution.

Dietary LC-PUFA in iron-deficient anaemic pregnant and lactating guinea pigs induce minor defects in the offsprings' auditory brainstem responses.

OBJECTIVES: We previously demonstrated that a mild pre-natal/early post-natal iron-deficient anaemic (IDA) diet devoid of long-chain polyunsaturated fatty acids (LC-PUFA) affected development, neurophysiology, and cerebral lipid biochemistry of the guinea pigs' progeny. Impacts of dietary LC-PUFA on altered cerebral development resulting from pre-natal IDA are unknown. To address this health issue, impacts of mild gestational IDA in the presence of dietary LC-PUFA on the offsprings' neural maturation were studied in guinea pigs using auditory brainstem responses (ABRs) and assessments of brain fatty acids (FAs). METHODS: Female guinea pigs (n = 10/group) were fed an iron sufficient (IS) or IDA diet (146 and 12.7 mg iron/kg, respectively) with physiological amounts of LC-PUFA, during the gestation and lactation periods. From post-natal day (PNd) 9 onwards, the IS + PUFA diet was given to both groups of weaned offspring. Cerebral tissue and offsprings' ABR were collected on PNd24. RESULTS: There was no difference in peripheral and brainstem transmission times (BTTs) between IS + PUFA and IDA + PUFA siblings (n = 10/group); the neural synchrony was also similar in both groups. Despite the absence of differences in auditory thresholds, IDA + PUFA siblings demonstrated a sensorineural hearing loss in the extreme range of frequencies (32, 4, and 2 kHz), as well as modified brain FA profiles compared to the IS + PUFA siblings. DISCUSSION: The present study reveals that siblings born from dams exposed to a moderate IDA diet including balanced physiological LC-PUFA levels during pregnancy and lactation demonstrate minor impairments of ABR compared to the control siblings, particularly on the auditory acuity, but not on neural synchrony, auditory nerve velocity and BTT.

17ß-estradiol induces stearoyl-CoA desaturase-1 expression in estrogen receptor-positive breast cancer cells.

BACKGROUND: To sustain cell growth, cancer cells exhibit an altered metabolism characterized by increased lipogenesis. Stearoyl-CoA desaturase-1 (SCD-1) catalyzes the production of monounsaturated fatty acids that are essential for membrane biogenesis, and is required for cell proliferation in many cancer cell types. Although estrogen is required for the proliferation of many estrogen-sensitive breast carcinoma cells, it is also a repressor of SCD-1 expression in liver and adipose. The current study addresses this apparent paradox by investigating the impact of estrogen on SCD-1 expression in estrogen receptor-α-positive breast carcinoma cell lines. METHODS: MCF-7 and T47D mammary carcinomas cells and immortalized MCF-10A mammary epithelial cells were hormone-starved then treated or not with 17ß-estradiol. SCD-1 activity was assessed by measuring cellular monounsaturated/saturated fatty acid (MUFA/SFA) ratios, and SCD-1 expression was measured by qPCR, immunoblot, and immunofluorescence analyses. The role of SCD-1 in cell proliferation was measured following treatment with the SCD-1 inhibitor A959372 and following SCD-1 silencing using siRNA. The involvement of IGF-1R on SCD-1 expression was measured using the IGF-1R antagonist AG1024. The expression of SREBP-1c, a transcription factor that regulates SCD-1, was measured by qPCR, and by immunoblot analyses. RESULTS: 17ß-estradiol significantly induced cell proliferation and SCD-1 activity in MCF-7 and T47D cells but not MCF-10A cells. Accordingly, 17ß-estradiol significantly increased SCD-1 mRNA and protein expression in MCF-7 and T47D cells compared to untreated cells. Treatment of MCF-7 cells with 4-OH tamoxifen or siRNA silencing of estrogen receptor-α largely prevented 17ß-estradiol-induced SCD-1 expression. 17ß-estradiol increased SREBP-1c expression and induced the mature active 60 kDa form of SREBP-1. The selective SCD-1 inhibitor or siRNA silencing of SCD-1 blocked the 17ß-estradiol-induced cell proliferation and increase in cellular MUFA/SFA ratios. IGF-1 also induced SCD-1 expression, but to a lesser extent than 17ß-estradiol. The IGF-1R antagonist partially blocked 17ß-estradiol-induced cell proliferation and SCD-1 expression, suggesting the impact of 17ß-estradiol on SCD-1 expression is partially mediated though IGF-1R signaling. CONCLUSIONS: This study illustrates for the first time that, in contrast to hepatic and adipose tissue, estrogen induces SCD-1 expression and activity in breast carcinoma cells. These results support SCD-1 as a therapeutic target in estrogen-sensitive breast cancer.

Mild iron deficiency anaemia during pregnancy and lactation in guinea pigs alters amplitudes and auditory nerve velocity, but not brainstem transmission times in the offspring's auditory brainstem response.

OBJECTIVES: It is well known that postnatal/early childhood iron deficiency (ID) anaemia (IDA) adversely affects infants' cognitive development and neurophysiology. However, the effects of IDA during gestation and lactation on the offspring are largely unknown. To address this health issue, the impact of mild IDA during gestation and lactation on the offsprings' neural maturation was studied in the guinea pig, using auditory brainstem responses (ABRs) latencies and amplitudes. METHODS: Female guinea pigs (n = 10/group) were fed an iron sufficient (ISD) or deficient diet (IDD) (144 and 11.7 mg iron/kg) during the gestation and lactation periods. From postnatal day (PNd) 9 onward, the ISD was given to both groups of weaned offspring. The offsprings' ABRs were collected on PNd24 using a broad range of stimulus intensities in response to 2, 4, 8, 16, and 32 kHz tone pips. RESULTS: Although the IDA siblings (n = 8) did not differ in brainstem transmission times (BTTs) compared to the IS siblings (n = 8), they showed significant delayed peak I latency at 100 and 80 dB, respectively. Additionally, significantly higher ABR wave amplitudes were observed in the IDA female offspring between 35 and 50 dB (4 kHz), a phenomenon suggestive of a neural hyperactivity (hyperacusis). DISCUSSION: In support to our previous findings, the present results indicate that a mild IDA during gestation and lactation can have detrimental effects on early development of the offsprings' hearing and nervous systems, particularly on neural synchrony and auditory nerve conduction velocity, but not on BTT.

Locomotor activity as an effective measure of the severity of inflammatory arthritis in a mouse model.

OBJECTIVE: Mouse models are valuable in preclinical studies of inflammatory arthritis. However, current methods for measuring disease severity or responses to treatment are not optimal. In this study a smart cage system using multiple sensors to measure locomotor activity was evaluated in the K/BxN serum transfer model of inflammatory arthritis. METHODS: Arthritis was induced in C57BL/6 mice with injections of K/BxN serum. Clinical index and ankle thickness were measured for 14 days. Locomotor activity was measured in smart cages for 23 h periods on Days 0, 7, and 13. The same measurements were taken in mice consuming diets supplemented or not with fish oil to evaluate a preventative treatment. RESULTS: Initiation, peak and resolution phases of disease could be measured with the smart cages. Locomotor activity including speed, travel distance, number of active movements and rear movements were all significantly lower on Days 7-8 of illness (peak) compared to Days 0 and 13-14 (resolution) (one-way repeated measures analyses, p<0.05). The clinical index and ankle thickness measurements did not capture differences between dietary groups. Significantly increased activity was measured in most of the locomotor parameters in the fish oil group compared to the control mice at both Days 8 and 14 (2-way repeated measures ANOVA, p<0.05). CONCLUSION: The measurement of locomotor activity provided a more detailed evaluation of the impact of inflammatory arthritis on animal well-being and mobility than that provided by measuring clinical index and ankle thickness, and could be a valuable tool in preclinical studies of inflammatory arthritis.