The dietary flavonoid eupatilin attenuates in vitro lipid peroxidation and targets lipid profile in cancer HeLa cells.

Eupatilin is a dietary flavonoid isolated from the alpine wormwoods, used for the genepy liqueur production. This flavone protects cells and tissues against oxidative stress and targets cancer cells, inducing cytotoxicity, cell circle arrest, apoptosis and mitochondrial dysfunction. This study examines the EUP in vitro antioxidant effects on cholesterol and phospholipid membrane oxidation and explores its ability to modulate the cancer cell lipid profile. This flavone remarkably protected fatty acids and cholesterol against oxidative degradation by scavenging lipoperoxyl radicals. EUP (24 h of incubation) significantly reduced viability and modulated the total lipid and fatty acid profiles in cancer HeLa cells. It induced marked changes in the phospholipid/cholesterol ratio, significant decreases in the levels of oleic and palmitic acids and a marked increase of stearic acid, involving an inhibitory effect on de novo lipogenesis and desaturation in cancer cells. Moreover, a noteworthy mitochondrial membrane depolarization, signs of apoptosis, abnormal mitosis with multi-nucleation (mitotic catastrophe) and morphological alterations were observed in cancer EUP-treated cells. Our results validate the EUP role as antioxidant agent for the treatment/prevention of disorders implicating a membrane lipid oxidative damage and substantiate cell lipid metabolism as another possible target of this dietary natural flavonoid in cancer HeLa cells.

Mugil cephalus roe oil obtained by supercritical fluid extraction affects the lipid profile and viability in cancer HeLa and B16F10 cells.

We explored the changes in viability and lipid profile occurring in cancer cells, murine melanoma cells (B16F10 cells) and human cervical carcinoma cells (HeLa cells), when exposed to 24 h-treatments with an n-3 PUFA-rich oil obtained by supercritical extraction with CO2 from Mugil cephalus processed roe (bottarga). The composition of the major lipid classes of bottarga oil was determined by the (13)C NMR technique. Reversed-phase HPLC with DAD/ELSD detection was performed to analyze cells' total fatty acid profile and the levels of phospholipids, total/free cholesterol, triacylglycerols, and cholesteryl esters. Cell-based fluorescent measurements of intracellular membranes and lipid droplets were performed on bottarga oil-treated cells using the Nile red staining technique. The treatments of cancer cells with bottarga oil reduced the viability and affected the fatty acid profile, with a significant n-3 PUFA increase in treated cells. Mullet roe oil uptake modulated the cancer cell lipid composition, inducing a remarkable incorporation of health beneficial n-3 PUFA in the polar and neutral lipid fractions. Bottarga oil treatment influenced the synthesis of intracellular membranes and accumulation of cytoplasmic lipid droplets in cancer cells.

Immunization with DISC1 protein in an animal model of ADHD influences behavior and excitatory amino acids in prefrontal cortex and striatum.

The Disrupted-in-schizophrenia 1 (DISC1) gene is involved in vulnerability to neuropsychiatric disorders. Naples high-excitability (NHE) rat model neuropsychiatric problems characterized by an unbalanced mesocortical dopamine system. Here, we assessed behavioral and neurochemical effects of immunization against multimeric rat DISC1 protein in adult NHE rats, an animal model of attention-deficit hyperactivity disorder and their Random-Bred (NRB) controls. Males of both lines received subcutaneous injections of vehicle (PB), adjuvant only (AD) or recombinant rat DISC1 protein purified from E. coli, suspended in AD (anti-DISC1) at age of 30, 45 and 60 postnatal days (pnd). At 75 pnd, the rats were exposed to a Làt maze and 2 days later to an Olton eight-arm radial maze, and horizontal (HA) and vertical activities (VA) were monitored. Non-selective (NSA) and selective spatial attention (SSA) were monitored in the Làt and in the Olton maze by duration of rearings and working memory, respectively. Post mortem neurochemistry in the prefrontal cortex (PFc), dorsal (DS) and ventral (VS) striatum of L-Glutamate, L-Aspartate and L-Leucine was performed. All immunized rats showed a clear humoral IgM (but not IgG) immune response against the immunogen, indicating that immunological self-tolerance to DISC1 can be overcome by immunization. NHE rats exhibited a higher unspecific IgM response to adjuvant, indicating an immunological abnormality. The sole anti-DISC1 immunization-specific behavioral in the NHE rats was an increased horizontal activity in the Làt maze. Adjuvant treatment increased vertical activity in both lines, but in the NRB controls it increased rearing and decreased horizontal activity. Liquid chromatography/tandem mass spectrometry analysis of soluble or membrane-trapped neurotransmitters aspartate, glutamate and leucine revealed increased soluble aspartate levels in the ventral striatum of NRB controls after anti-DISC1 immunization. Immune activation by adjuvant independent of simultaneous DISC1 immunization led to other specific changes in NHE and control NRB rats. In DISC1-immunized NHE rats, horizontal activity in Lat maze correlated with membrane-trapped glutamate in PFc and in the NRB rats, duration of rearing in Olton maze correlated with membrane-trapped glutamate in PFc and aspartate in dorsal striatum. In addition to non-specific immune activation (by AD), the postnatal anti-DISC1 immune treatment led to behavioral changes related to mechanisms of activity and attention and had influenced amino acids and synaptic markers in striatum and neocortex in the adult NHE as well as control animals.

Encapsulation and modified-release of thymol from oral microparticles as adjuvant or substitute to current medications.

The aim of this study was to encapsulate, thymol, in natural polymers in order to obtain (i) taste masking effect and, then, enhancing its palatability and (ii) two formulations for systemic and local delivery of herbal drug as adjuvants or substitutes to current medications to prevent and treat several human and animal diseases. Microspheres based on methylcellulose or hydroxypropyl methylcellulose phthalate (HPMCP) were prepared by spray drying technique. Microparticles were in vitro characterized in terms of yield of production, drug content and encapsulation efficiency, particle size, morphology and drug release. Both formulations were in vivo orally administered and pharmacokinetic analysis was carried out. The polymers used affect the release and, then, the pharmacokinetic profile of thymol. Encapsulation into methylcellulose microspheres leads to short half/life but bioavailability remarkably increases compared to the free thymol. In contrast, enteric formulation based on HPMCP shows very limited systemic absorption. These formulations could be proposed as alternative or adjuvants for controlling pathogen infections in human or animal. In particular, methylcellulose microspheres can be used for thymol systemic administration at low doses and HPMCP particles for local treatment of intestinal infections.

Effect of free thymol on differential gene expression in gastric mucosa of the young pig.

Thymol is the most common molecule in thyme and has been proposed as an oral alternative to antibiotics in the feed of pigs and broilers. The knowledge of the in vivo physiological effects of thymol on tissues is limited, particularly its impact on the gastric mucosa, where it is primarily absorbed when it is orally supplied. In this study, thymol (TH, 50 mg/ kg BW) or a placebo (CO) was introduced directly into the stomach of 8 weaned pigs that were slaughtered 12 h later and sampled for gastric oxyntic and pyloric mucosa. The analysis of whole transcript expression was performed using Affymetrix© Porcine Gene 1.1 ST array strips. Affymetrix Transcripts IDs were associated with 13 406 human gene names based on Sus scrofa Ensemble. Gene Set Enrichment Analysis was performed, comparing TH and CO pigs. For each gene set, the normalized enrichment score (NES) was defined as significant when the false discovery rate % was <25 and the P-value of NES was <0.05. In response to TH, 72 and 19 gene sets were significantly enriched in the oxyntic and pyloric mucosa, respectively. Several gene sets involved in mitosis and its regulation ranked near the top, primarily in the oxyntic mucosa; the gene set DIGESTION ranked first and ninth in the pyloric and oxyntic mucosa, respectively. Within this group, somatostatin (SST), SST receptors, peptide transporter 1 (SLC15A1) and calpain 9 (gastrointestinal tract-specific calpain) were the most strongly upregulated genes. Thymol reduced the enrichment of 120 and 59 gene sets in the oxyntic and pyloric mucosa, respectively. Several gene sets related to ion transport and channeling and aqueous pores across membranes, including short transient receptor potential (TRP) channel 4, potassium voltage-gated channel members 1 and 2, and ryanodine receptors 2 and 3, were less enriched. The downregulation of these genes sensitive to thymol in vitro could depend on the thymol dose and contact with the gastric tissues that causes an adaptive response with their reduced activation. Conversely, the activation of the TRPA1 gene (ranked 1072 and 128 among all the genes in the oxyntic and pyloric mucosa, respectively) indicates the involvement of another TRP-regulating cellular calcium storage. In conclusion, the stimulation of gastric proliferative activity and the control of digestive activity by thymol can influence positively gastric maturation and function in the weaned pigs. These properties should be considered in addition to thymol's antimicrobial properties when supplementation of this molecule in feed is evaluated.

The human ALL-1/MLL/HRX antigen is predominantly localized in the nucleus of resting and proliferating peripheral blood mononuclear cells.

The ALL-1 gene is an important regulator of embryonal and hematopoietic development, and structural variants of the human gene generated by chromosomal translocations and other genomic alterations presumably act as oncogenes in the pathogenesis of acute leukemias and other hematological malignancies. Antisera against two different epitopes of the human ALL-1 protein (anti-ALL1-N and anti-ALL1-C) were produced. Both sera revealed indistinguishable patterns of antigen localization in human peripheral blood mononuclear cells (PBMCs). In resting PBMCs, the antigen was distributed in a speckled pattern across the nuclei, with an increased density at the nuclear envelope and the nuclear indentation. In mitotically stimulated PBMCs, the antigen surrounded the condensing chromosomes but did not colocalize with chromatin or the nuclear scaffold. The antigen is considered a marker for a novel nuclear subcompartment, a perichromosomal area termed the "chromosomal envelope." In Western blot experiments, the anti-ALL1-N serum reacted with a polypeptide corresponding to the expected full-length 430-kDa ALL-1 protein. Recombinant proteins representing the AT-hook and zinc binding subdomains of the ALL-1 protein interacted in vitro with a degenerate mixture of double-stranded oligodeoxynucleotides. Thus, the ALL-1 protein probably is a DNA-binding protein with both a sequence-unspecific (AT-hook) and a sequence-specific (zinc binding subdomains) double-stranded DNA binding mode.