Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade α-1,3-glucan and allow remodeling of the cell wall, α-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal α-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1  increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall α-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl α-1,3-glucan , used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates , showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1Δ strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71), showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal GH-71 into at least five groups, for which specific conserved sequences can be identified.α-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y) form of  Paracoccidioides brasiliensis complex  is a human systemic mycosis caused by four species, comprised by the Schizosaccharomyces pombe, P. brasiliensis, H. capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans and Aspergillus species S. pombe demonstrate that during vegetative growth, the cell wall α-1,3-glucan is built with two linear glucose polymers, each 260 residues-long, interconnected through α-1,3 and α-1,4 glycosidic linkages P. brasiliensis, α-1,3-glucan is composed of a single linear polymer of α-1,3 linked- glucose residues, and occasional ramifications of one glucose moiety bound to the backbone by α-1,4 linkages α-1,3-Glucan has been found in a few fungal species such as Streptococcus mutansα-1,3-Glucanases (EC 3.2.1.59), also called mutanases due to their ability to degrade the extracellular glucan synthesized by the bacterium S. pombe, two α-1,3-glucanase genes are present (agn1 and agn2), whose translation products Agn1p and Agn2p are involved in different cell processes. Agn1p is involved in cytokinesis S. pombe agn1 mutants are unable to separate as free cells, impairing the physical division of the cell during cell fission S. pombe agn2 mutants In A. nidulans α-1,3-glucanase is secreted to the cell wall and mobilizes α-1,3-glucan, the main reserve material accumulated during vegetative growth in the cell wall; once monosaccharides are released, they are captured and metabolized by the cell during starvation Trichoderma harzianum, α-1,3-glucanase degrades cell wall of plant pathogenic fungi, thus becoming an inhibitor of spore germination and mycelial growth of a wide range of fungal pathogens After the exhaustion of glucose, P. brasiliensis, we aimed to characterize the P. brasiliensis α-1,3-glucanase by heterologous expression of its encoding gene, AGN1, and purification of its transcriptional product, Agn1p. Functionality of the gene was assessed by complementation of an S. pombe agn1Δ mutant with the P. brasiliensis AGN1 gene.As a further step into the comprehension of the cell wall α-1,3-glucan metabolism in P. brasiliensis Pb73 (ATCC 32071), was grown on liquid YPD , either at 23°C (M cultures) or 37°C (Y cultures) with or without 5% horse serum (Gibco) with continuous shaking at 100 rpm for 3–4 days. Escherichia coli QIAGEN EZ chemically competent cells , used for propagation of plasmids and cloning experiments was grown in Luria–Bertani (LB) medium  and supplemented with 100 µg/ml ampicillin  when required for plasmid selection. E.coli M15 [pREP4] , used for heterologous expression and Agn1p purification, was grown in LB medium with 25 µg/ml kanamycin  and supplemented with 100 µg/ml ampicillin  for plasmid selection. Schizosaccharomyces pombe, strains wt-64  and 1252  S. pombe agn1 mutant), Edinburgh minimal medium (EMM) was employed P. brasiliensis using TRIzol Reagent™ , following the manufacturer's instructions. S. pombe gDNA from wild type strain wt-64, or plasmid DNA from S. pombe strain 1252, were isolated according to Hoffman and Winston Genomic DNA (gDNA) extraction was performed as previously described P. brasiliensis AGN1, a HindIII partial genomic library was constructed as follows: 100 µg of P. brasiliensis DNA were digested with HindIII (Invitrogen), and size-fractionated fragments  were cloned into pBluescript SK vector (Stratagene). Resulting transformants were collected and screened by colony hybridization, with a 750 bp PCR amplified fragment of the putative H. capsulatum AGN1 gene, by using Mut(F): 5′ ATY GAY GCA TTY GCW CTY AAY 3′ and Mut (R): 5′ GAY TCR CCG TAG TC 3′, primers. A positive clone yielding plasmid pMP1, containing a 2.3 kb insert was isolated and sequenced, showing to contain a partial sequence of the gene. The complete P. brasiliensis AGN1 gene sequence was obtained using the SMART™ RACE cDNA amplification kit . The following primers were used as gene-specific primers: HV1 (5′-GTA CCA GAA TGT GAT AAT GTC GGC GG-3′) and HV2 (5′-GCT GGA CAA ATT CTG GCT GTA GTG TG-3′) towards the 5′ region and GlnF (5′ AGT TTT GGG TCA TAA GCC G 3′) towards the 3′ region. cDNA was amplified by RT-PCR, and the amplicons cloned into vector pDrive  for sequencing. For sequencing reactions, plasmid preparations were carried out with the Concert™ Miniprep System  or Axyprep Plasmid Miniprep Kit . Nucleotide sequencing was automated on an ABI PRISM 377 DNA sequencer (Perkin Elmer) (Unidad de Estudios Genéticos y Forenses (UEGF), Centro de Microbiología y Biología Celular, IVIC, Caracas, Venezuela). The sequence has been deposited in GenBank (accession number EF679780).For isolation of http://www.vivo.colostate.edu/molkit/hydropathy/. The genome database of P. brasiliensis (http://www.broadinstitute.org/) was used to verify the presence of one or more genes belonging to the family 71 of glycoside hydrolases. The MEGA 4 software was used for sequence alignment  and the construction of phylogenetic tree was done by the neighbor-joining method.Assembly of the nucleotide sequences and translated protein sequences were generated with the Vector NTI suit package . Homology searches were performed on the GenBank database using BLAST 2.0 AGN1, primers RT3∶5′-GCA GCA AGT TAT CAC ACT AC-3′ and RT4∶5′-TGG TTC CGT CAT ACA TTT TA-3′ were used. For expression analysis of AGS1, sequence specific primers FrwAGS1_RT: 5′-AAA TGC GGC ACG GAG GAG A-3′ and RevAGS1_RT: 5′-AAG GGT GGT ATC AAG TGC CGA GT-3′ were used. To find the best internal control as normalizer for the expression experiments, two genes were used. Amplification of 18S rRNA was carried out, using the primers 18S S3∶5′-CGA TTC CGG AGA GGG AGC C- 3′ and 18S AS3∶5′-CGT ATC GGG ATT GGG TAA TTT GC-3′. A second reference gene (Pbl34) which has no changes in transcription on both morphologies 18S gene were observed. Therefore, the Pbl34 gene was chosen as the normalizer gene for all subsequent experiments. Quantitative PCR was performed in triplicate on an iQ5 real time PCR detection system, using the GoTaq® qPCR Master Mix , in a 15 µl volume . Reaction conditions were as follows: 95°C for 3 min, followed by 40 cycles at 94°C for 10 s, 58°C for 30 s, and 72°C for 30 s, with dissociation conditions of 95°C for 1 min, 55°C for 1 min, and 81 cycles starting at 55°C, with temperature increases of 0.5°C every 10 s up to 95°C. PCRs with serial dilutions of P. brasiliensis cDNA as template were used to calculate the amplification efficiency for each pair of primers. All Ct values were normalized to the Ct values of the standard gene and the relative expression levels were calculated using the 2−ΔΔCT method Total RNA was treated with DNase by using the TURBO DNA free™ kit . The RETROScript™ kit  was used for reverse transcription of mRNA. For real-time PCR of P. brasiliensis AGN1 ORF (Gen-Bank Accession No. EF679780) deprived of the sequence coding for the putative signal peptide was PCR-amplified from cDNA, using primers HVC2 5′-CAT AGA GCT CAT TCA AAC ATC CAC GCT-3′ and HVC3 (5′-GGA TCC AAG GCT GTA TTT GCC CAT TTC-3′), and cloned at the SacI and BamHI restriction sites of plasmid pQE30Xa (Qiagen), generating pHV2. E.coli M15 [pREP4]  was transformed with pHV2 or pQE30Xa empty (as negative control), grown on LB medium supplemented with 100 µg/ml ampicillin  and 25 µg/ml kanamycin  at 37°C, following the manufacturer’s indications. For protein expression experiments, each transformant was grown on LB medium supplemented with 100 µg/ml ampicillin , 25 µg/ml kanamycin , 500 mM NaCl, 0.2% glucose and 1 mM sorbitol at 37°C for 5h until culture OD600 reached 0.7. Protein expression was induced with the addition of 0.5 mM IPTG and cultures were grown at 23°C overnight 2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Cell pellets were resuspended in lysis buffer supplemented with 1 µl/ml protease inhibitor cocktail (Sigma P-8215), treated with 1 µg/ml lysozyme (Sigma L-6876) on ice for 30 min. Cells were lysed with 0.17 µm glass beads 2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Agn1p-his was eluted in fractions by addition of elution buffer . The eluates were pooled and concentrated to 1 ml using Amicon ® Ultra-4 (Millipore) with a 30 kDa cutoff. The concentration process was performed at 4000g for 90 min at 4°C. Purity was monitored by SDS-PAGE analysis employing Mini-PROTEAN chambers ® II Electrophoresis Cell , as recommended by the manufacturer and according to the size of the expected product For Agn1p-his purification, the P.brasiliensis Agn1p, P. brasiliensis yeast-like cell wall was extracted by alkaline separation as in In order to test the enzymatic activity and specificity of −1, using a Nicolet iS10 IR spectrometer , coupled to the OMNIC 8.0 software.Samples were prepared as KBr pellets. IR spectra were recorded from 3500 to 500 cm13C-NMR experiments were carried out either in a Bruker 300 or Bruker 500 Ultrashield spectrometers at 75 MHz and 125 MHz. The polysaccharides (20 mg) were dissolved in D2O (1 ml) using a data collection time of 16 h 3COONa buffer  in a final volume of 1 ml. Reactions were stopped by heating at 100°C All reactions were carried out with 100 µg of Agn1p-his and 1 mg/ml of SCMG in CH3COONa buffer pH 5.0 were preincubated on ice for 15 min with Agn1p-his or glucoamylase (0.5 µg/ml) (Roche 1202332), as a positive control of inhibition. Remaining Agn1p-his activity was measured by incubation with SCMG (1 mg/ml) in optimal conditions . Remaining glucoamylase activity was measured in 50 mM sodium acetate, pH 5.6, containing 1 mg/ml of starch  To test the effect of inhibitors, 1-deoxynojirimycin , D-glucono-1,5-lactone , or 50 mM CHSubstrate specificity was determined by incubation at optimal conditions of Agn1p-his with substrates at a final concentration of 1 mg/ml. Carboxymethylated α--glucan (SCMG), starch , carboxymethyl-chitin , carboxymethyl-laminarin , carboxymethyl-cellulose , and dextran T500 (Pharmacia), were used as substrates. Reaction products were analyzed colorimetrically 4 1.5 g; K2CO3 10 g, 1.25 ml NaOH 10% in 200 ml of distilled water) using the spray-type sprayer Flask Aldrich (Z190373 -1EA). Plates were left to dry and developed for about 1 h at 60°C, producing yellow spots on a pink background The enzymatic reactions were concentrated to 50 µl by lyophilization. Aliquots of 5 µl were placed on a TLC plate , using propanol/water  as the mobile phase, for three hours in a closed chamber previously saturated with the solvent mixture. As standard markers, glucose , maltose , maltotriose  at a concentration of 333 µg/ml were placed, in separate contiguous lanes. After completion of the run, the plates were dried at 60°C for 10 min, and impregnated with iodine vapors or a specific staining solution for carbohydrates  as described in Suga and Hatakeyama agn1  and pREP3X  were used. Transformants were evaluated by PCR using the primers listed in Both plasmids were used to transform S. pombe 1252 by the P. brasiliensis AGN1 gene was followed by observation of calcofluor white stained cells in a fluorescence microscope Leica DM2000 equipped with H3 filter. Photographs of fluorescent images were taken with a Leica DFC310 FX digital camera, using an immersion objective with 100X magnification. For microscopic observation, 50 µl of cell suspension was mixed with 50 µl of 1 µg/µl calcofluor white . The mixture was smeared onto slides plates pre-treated with 20 µl of 0.1% polylysine, air-dried, and washed with PBS. To quantify the degree of complementation, sedimentation assays were performed as in Complementation of P. brasiliensis AGN1 gene has three exons that account for a putative coding region of 1495 bp, separated by two introns, all confirmed by comparison of the sequence of the RT-PCR product with the corresponding genomic sequence .The sequence . It encosequence , with hiS. pombe and A. nidulansIn silico analysis of the deduced protein shows a signal peptide corresponding to the 21 initial amino acids, and a main domain homologous to the GH-71 family, which extends from residues 23 to 432 , similarP. brasiliensis genome database (http://www.broadinstitute.org/) shows that AGN1 is the only gene in the P. brasiliensis genome related to the hydrolysis of α-1,3-glucan. A Clustal analysis was performed that included 90 complete amino acid sequences of fungal glucanases present in GenBank and CAZy databases (http://www.cazy.org/GH71_eukaryota.html), grouping P. brasiliensis Agn1p into the glycoside hydrolase family 71  has been reported as a booster for α-1,3-glucan synthesis in P. brasiliensis α-glucanase (Agn1p) was performed, using E. coli as the expression host. The cDNA without the signal peptide coding sequence was cloned into the pQE30Xa plasmid in frame with the His-tag present in the commercial plasmid, to produce the pHV2 vector. Induction of protein expression was obtained by addition of IPTG and the protein purified by affinity chromatography. A SDS-PAGE of the purified Agn1p-His showed a single band with an estimated molecular mass of 51.8 kDa  and the presence of glucose residues linked by α-1,3 bonds (∼918 and 840 cm−1) (data not shown). 13C-NMR clearly showed the corresponding signal of carbonyl groups , chemically modified from purified analysis . IR pattα--glucan (SCMG) when tested against a battery of glucose or glucosamine polymers  (P. brasiliensis Agn1p were established at 1 h as pH 5.0 and 40°C (not shown). No inhibitory effect was observed upon Agn1p-his pre-incubation with inhibitors of exo-catalytic hydrolases  (2 = ∼0.9786) were the main hydrolysis products.Agn1p was only active against dextran) . Optimallactone) . Endo-calactone) , where hS. pombe agn1 null mutant strain 1252: (a) pHV3, containing the complete P. brasiliensis AGN1 gene, including its original signal peptide coding region (S. pombe strain HLVSP3), and (b) pHV4, which includes a chimeric P. brasiliensis AGN1, whose signal peptide coding region was substituted by the S. pombe agn1 signal peptide coding region, constructed by PCR overlap extension (S. pombe strain HLVSP4). As positive control, S. pombe HLVSP5, containing plasmid pREP3X-+agn1, which includes the complete ORF from the +S. pombe agn1 gene, was used. Negative control consisted of S. pombe 1252 transformed with the empty vector pREP3X, (HLVSP6 strain). Cells were analyzed by calcofluor white staining, confirming that the strains carrying the +agn1 and AGN1 ORFs were able to suppress the separation defect shown by the S. pombe 1252 mutant  interrupted by two introns (accession number EF679780), whose product, Agn1p, is 77% identical to other fungal α-1,3-glucanases. P. brasiliensis Agn1p has a molecular mass of ∼51 kDa after SDS/PAGE; according to its amino acid sequence it can be classified into the poorly characterized family 71 of glycoside hydrolases (GH-71). A clustal alignment of P. brasiliensis Agn1p with other fungal GH-71 allows us to infer the location of 5 conserved residues, specifically aspartic and glutamic acids (D and E respectively), which may correspond to amino acids involved in the acid-base catalytic mechanism P. brasiliensis Agn1p clustering into G1. G3, G4 and G5 share the conserved sequence (T/S)WND, while G4 and G2 shared the consensus sequence: FALN. It should be noted that glucanases that exhibit a mutan-binding domain (MBD) are grouped exclusively within the group G4 , showing high identity to T. harzianum MBD GD, GESHYI, YMAPVS, KNWVF for group G2; T(F/I)EG for group G3; GIDAFALNIG, F(A/V)SF, SKNW, (I/V)YWYR, G(I/L)YNFN for group G4, (S/N)(L/F)D(M/V), F(A/V)LN for group G5.P. brasiliensis Agn1p are predicted to be a signal peptide, suggesting the location of Agn1p towards the P. brasiliensis membrane or cell wall, where the α-1,3-glucan, a virulence factor and the specific substrate for Agn1p, is located. This location is shared by most of the fungal α-1,3-glucanases so far studied P. brasiliensis, H. capsulatum, A. fumigatus, C. immitis, for proteins that bind to the extracellular matrix Candida albicans cell wall proteins involved in cell adhesion processes. Despite those possible post-translational modifications, we were able to achieve the purification of functional Agn1p from heterologous expression in E. coli, showing that in the absence of post-translational modifications (due to intracellular heterologous expression) the glucanase activity remains, as was recently reported for a recombinant glucanase from T. harzianum expressed also in E. coliP. brasiliensis α-1,3-glucanase, measured at optimal conditions with SCMG as the soluble substrate, had a specific activity of 0.075 U/mg. It should be noted that the conditions used for carboxymethylation have been described as adequate to ensure solubility without alteration of the linear structure of the polysaccharide 13C-NMR  spectra of SCMG indicate that carbonyl groups were properly added to the otherwise unchanged polysaccharide, data that support the effectiveness of the reaction, and the maintenance of an α anomeric configuration in the resulting SCMG P. brasiliensis Agn1p enzyme showed high specificity for its proposed natural substrate, cell wall α-1,3-glucan , surrounded by a secondary septum , through which septum degradation and cell separation starts. Therefore, agn1 mutants are incapable of splitting, as shown with calcofluor white staining  gene recently reported P. brasiliensis, and found to contribute to pathogenesis in H. capsulatum by concealing immunostimulatory β-glucans from detection by host phagocytic cells P. brasiliensis α-1,3-glucan , and the fact that this polysaccharide is absent from the natural fungal host, leads us to propose both, its mechanisms of synthesis (by blocking it) and degradation (by stimulating it) as potential targets for the development of specific drugs against P. brasiliensis, which might result in the depression of fungal virulence, and allow the action of the natural immune response of the infected organism against the fungus.The fact that Figure S1AGN1 genomic sequence (gDNA) from P.brasiliensis strain Pb-73. Highlighted in yellow, the deduced amino acid sequence of P. brasiliensis α-1,3-glucanase Agn1p. Highlighted in italics and bold, the putative start codon and the methionine residue attached, respectively. In red letters, 21 amino acids belonging to a putative signal peptide. In green, AGN1 intron sequences, (their processing sites are underlined). Post-translational putative modification sites are highlighted in colored boxes: blue: cell adhesion; purple, N-glycosylation.(TIF)Click here for additional data file.Figure S2 13C-NMR spectra.SCMG The box indicates the location of the signals corresponding to the carbonyl group, while the arrows point to the signature band of the α-1,3 configuration of both SCMG and α-1,3-glucan.(TIF)Click here for additional data file.


The expression levels of tight junction occludin and ZO-1 were decreased (p < 0.05). The reduced fluorescence of targets and change of cellular morphology were recorded. The tight junction occludin and ZO-1 mRNA expression linearly declined with increasing β-conglycinin (p < 0.001).Soybean allergy presents a health threat to humans and animals. The mechanism by which food/feed allergen β-conglycinin injures the intestinal barrier has not been well understood. In this study, the changes of epithelial permeability, integrity, metabolic activity, the tight junction (TJ) distribution and expression induced by β-conglycinin were evaluated using IPEC-J2 model. The results showed a significant decrease of trans-epithelial electrical resistance (TEER) ( SoybeanThe intestinal mucosa represents a crucial border between the organism and its environment. The large contact surface allows efficient nutrient absorption, acts as an important barrier for pathogens and toxins, and participates in the innate immune response ,6. As rePiglets easily suffer from soybean-induced allergy, so the presence of allergenic components including intact β-conglycinin and its enzyme-hydrolyzed peptides greatly restricts their use in neonatal and weanling pigs. Soybean allergies usually cause inflammatory disorders in the small intestine, characterized by villous atrophy and hyperplasia in the crypt, as well as accelerated enterocyte proliferation, apoptosis and migration –18. Howein vivo situation. Meanwhile, it could also give some related hints for soybean-allergic patients due to porcine physiological and immunological similarity to human beings [The intestinal porcine epithelial cells originated from jejunum (IPEC-J2) retained most of their original epithelial nature . So we on beings ,20. In t2.2.1.p < 0.001, R2 = 0.919) levels at 24 h. The highest concentration of β-conglycinin (3 mg/mL) reduced the TEER to 67% of the control after 24 h, respectively. The maximal reduction of TEER (versus control) was 38% after 72 h incubation, respectively.As shown from 2.2.R2 = 0.862, p < 0.001) at 24 h (versus control) after 72 h incubation, respectively.A significant linear reduction of cellular metabolic activity was detected after application of 0.5–3 mg/mL β-conglycinin .Detection of AP activity as a marker for enterocyte differentiation  showed a2.4.p < 0.05).The tight junction proteins (occludin and zonula occluden (ZO)-1) were located at the cell-cell contact regions, as shown in 2.5.R2 = 0.941, p < 0.001; ZO-1, R2 = 0.956, p < 0.001) from 0.5–3 mg/mL (versus control) and ZO-1 (versus control) was 57% and 59% for β-conglycinin.Tight junction occludin or ZO-1 mRNA expression was assessed after 24 h exposure to β-conglycinin of different levels. The tight junction mRNA expression tended to linearly decline with increasing β-conglycinin  could increase the intestinal permeability –34. TEERet al. (2010) [The MTT assay is a standard method for the detection of metabolic activity in cell culture, which indirectly demonstrates the damage of membrane function. The present data show that β-conglycinin reduces the cellular metabolic activity in a dose-dependent manner, suggesting the reduction of cellular activity, which is similar to Xu . (2010)  using th. (2010) –16,39. I. (2010) . The aboIntestinal AP has an essential function in maintaining epithelial integrity, whose loss may increase the permeability to inflammation and sepsis ,41. In oTJ, a major mode of cell-to-cell adhesion, is a key determinant of intestinal barrier function preventing bacteria, endotoxin and toxicity macromolecule from crossing an epithelial sheet between adjacent cells. It is organized by specific interactions between various intracellular proteins  and transmembrane proteins  . The redThe regression analysis suggests that β-conglycinin has a linear relationship with the cellular permeability, metabolic activity, integrity, and TJ gene expression. This means that the increasing soybean allergens induce the tight junction to loosen, and then augment the epithelial permeability. Moreover, from observation, it seems that indexes of the current study change seriously with the extension of incubation time.4.4.1.et al. (1987) [β-Conglycinin was isolated from defatted soy flour by the method of Setsuko . (1987) . The glo4.2.2 . The culture medium was changed every other day.The porcine intestinal epithelial cells from jejunum (IPEC-J2) were kindly donated by Guoyao Wu of China Agricultural University. The IPEC-J2 were seeded in cell culture flasks and cultured in DMEM/F12 medium , supplemented with 10% fetal bovine serum , 1% Penicillin-Streptomycin , and 1% glutamine  at 37 ºC in a humidified atmosphere of 5% CO4.3.4/cm2. When monolayer of cells was completely differentiated, cells were treated with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/mL of β-conglycinin. The TEER was measured using the millicell-ERS resistance system  after incubation for 24, 48, and 72 h, respectively.IPEC-J2 were seeded on the millicell membrance  cell culture inserts  at a density of 5 × 104.4.Metabolic activity was measured by MTT assay. IPEC-J2 were then treated with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/mL β-conglycinin for 24, 48, and 72 h, respectively. Exactly 20 μL of MTT solutions  was added to each well and incubated for 4 h at 37 ºC. The mixture reaction was carefully taken out and 150 μL dimethyl sulfoxide  was added to each well, which was allowed to stand 10 min to allow it to completely mix. After the crystals were fully dissolved, the optical density was measured at 570 nm by Microplate Reader .4.5.Cellular membrane integrity was assessed by measurement of alkaline phosphatase (AP) activity in the supernatant. Cells were grown in a 96-well microplate and treated with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/mL β-conglycinin for 72 h, respectively. The AP activity of the incubation medium was measured by the AP Test kit .4.6.IPEC-J2, grown on cover glass within six well plates  for complete confluence, were treated with 0.5 mg/mL β-conglycinin media for 24 h. After rinsing in PBS three times, samples were fixed with cold acetone for 15 min at room temperature. Next, cells were permeabilized with 0.5% Triton X-100 for 5 min and blocked with 5% Bovine Serum Albumin (BSA) for 30 min at 37 ºC. After washing, samples were incubated with rabbit-anti-occludin or rabbit-anti-ZO-1  as the primary antibody for 30 min at 37 ºC then with FITC-conjugated goat-anti-rabbit  as the second antibody for 2 h at room temperature. The views for each sample were captured using laser scanning confocal microscope  and representative images were presented.4.7.g for 30 min at 4 ºC. Protein quantification of supernatant was determined by method of BCA kit .IPEC seeded in cell culture flasks were treated with 0.5 mg/mL β-conglycinin media for 24 h, then cells were washed with PBS (0.01 M). Every flask of samples was lysed on ice with a 500 μL cell lysis buffer  and supplemented with protease inhibitors  for 30 min. Sample collections were sonicated three times for 20 s and centrifuged at 10,000× Exactly 12 μg of cell total proteins extraction was separated by SDS-PAGE and transferred onto PVDF membranes. The rabbit anti-occludin, rabbit anti-ZO-1 or rabbit anti-β-actin  antibodies were used as the primary antibody. The horseradish peroxidase-conjugated anti-rabbilt IgG  was incubated as the second antibody. The Luminata Crescendo Western HRP Substrate  was applied for chemiluminescent detection using tabletting and autoradiographic film . Grey levels were estimated using Quantity One software-4.6.2 (Basic) . Western blot band density was compared to β-actin in each lane as a loading control.4.8.2+ and Mg2+, harvested, centrifuged, and subjected to RNA extraction procedure.The IPEC-J2 was stimulated with varying concentrations of β-conglycinin  for 24 h. Then the cells were rinsed twice with phosphate-buffered saline (PBS) without CaTotal cellular RNA was extracted from IPEC-J2 with E.Z.N.A. Total RNA Kit II  according to the manufacturer’s instructions. To prevent DNA contamination, DNase digestion was performed with RNase-free DNase. The Purified RNA extracts were stored at −80 ºC in RNase, DNase-free water  until use. The concentration and purity of RNA extracts was determined using the absorbance at 260 and 280 nm in a spectrophotometer . The samples with the ratio 260 nm/280 nm between 1.9 and 2.1 were further evaluated for their quality. The integrity of RNA was determined on 1% agarose gels and visualization of the 28S and 18S ribosomal RNA.4.9.2O to complete the final volume. The reserve transcription mix was incubated at 25 ºC for 10 min, heated to 37 ºC for 120 min, and finally inactivated at 85 ºC for 5 min. The resultant cDNA was stored at −80 ºC until use.Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kits . The reverse transcription was carried out in a 20 μL final volume that included 2 μL 10× RT Buffer, 0.8 μL 25× dNTP Mix (100 mM), 2 μL 10× RT Random Primers, 1 μL MultiScribe™ Reverse Transcriptase , 2 μg RNA template, and Nuclease-free H4.10.2O. The 40 thermal cycles of 2 min at 50 ºC, 10 min at 95 ºC, 15 s at 95 ºC and 1 min at 60 ºC were utilized according to manufacturer recommendations.The real-time quantitative PCR was carried out using a StepOne Plus real-time PCR system . TaqMan Gene Expression Master Mix was used according to manufacturer’s specifications . Primer and probe sets for target genes were prevalidated in TaqMan Gene Expression Assay kits (ABI): . The PCR reaction was carried out in 96-well reaction plates with 10 μL TaqMan Gene Expression Master Mix (2×), 1 μL TaqMan Gene Expression Assay (20×), 2 μL cDNA template and 7 μL Nuclease-free H−ΔΔCt method. Target gene expression was normalized to β-actin messenger RNA (mRNA) levels.Relative quantification of target genes was calculated using the 24.11.r) was used for jointly distribution variables. Statements of statistical significance were based upon p ≤ 0.05.Data of relative gene expression were analyzed using the general linear model procedure of Statistical Package for Social Sciences version 11.5 . Differences among means were tested using Duncan’s multiple range tests. Linear regression with calculation of the correlation factor (5.The altered TJ expression induced by β-conglycinin gives rise to the barrier dysfunction of intestinal epithelium.


The DPPH scavenging (IC50) activity varied between 2.52 ± 0.03 mg/mL and 3.29 ± 0.01 mg/mL and FRAP ranged from 7.39 ± 0.08 to 104.2 ± 6.34 μmol TE/g DW. Among all the measured micro- and macrominerals K content was the highest followed by N, Na, Ca, Mg, P, Fe, Zn, and Mn. The overall findings proved that ornamental purslane was richer in antioxidant properties, whereas common purslane possesses more mineral contents than ornamental ones.The methanolic extracts of 13 accessions of purslane were analyzed for their total phenol content (TPC), total flavonoid contents (TFC), and total carotenoid contents (TCC) and antioxidant activity of extracts was screened using FRAP assay and DPPH radical scavenging methods. The TPC, TFC, and TCC ranged from 0.96 ± 0.04 to 9.12 ± 0.29 mg GAE/g DW, 0.13 ± 0.04 to 1.44 ± 0.08 mg RE/g DW, and 0.52 ± 0.06 to 5.64 ± 0.09 mg ( Portulaca oleracea has been reported to be the richest vegetable source of omega-3 (ω-3) fatty acids (FA) yet examined [ω-3 fatty acids and traces of 22: 5ω-3 and 22: 6ω-3 [Antioxidants are vital substances which possess the ability to protect the body from damages caused by free radical-induced oxidative stress, whereas the micro- and macrominerals are the key components for overall body growth and development. Purslane has been ranked as the eighth most common plant in the world  and is lexamined . Subsequ22: 6ω-3 , 11. Pur22: 6ω-3 . The abu22: 6ω-3 , 10. In 22: 6ω-3 , 13. Pur−1 soil. Soil nutrient status was 0.16% total N, 5.65 ppm available P, 15.3 ppm available K, 3295 ppm Ca, and 321 ppm Mg. At field capacity, soil water retention was 31.18% (wet basis) and 45.31% (dry basis). The experimental soil belongs to the Serdang series.The experiment was conducted during May 2012 to September 2012 in a glasshouse at the Faculty of Agriculture, University Putra Malaysia , and Food Biotechnology and Functional Food Research Laboratory, Faculty of Food Science and Technology, UPM, Malaysia. The plastic pots (24 × 22 cm) were filled with soil  of pH 4.8 with 2.6% organic carbon, 1.24 g cc-1 bulk density, and CEC of 7.07 me 100 gThirteen (13) samples of 10–15 days young seedlings of purslane were collected from different locations of West Peninsular Malaysia considering location and morphological variation of the plants and transplanted into the pots with prepared soils organized in a randomized complete block design with three replications. Brief descriptions of the collected samples and locations are shown in Four to five seedlings were transplanted in each pot and were surface-irrigated thrice a week  throughout the growing period using only tape water. Sixty day-old matured harvested purslane samples were divided into two parts of each accession: one for the analysis of antioxidant properties and the other for mineral analysis. Due to light and temperature sensitivity of antioxidant properties the fresh samples were oven-dried at 45–50°C for one week then ground and stored in −20°C freezer until analysis. And for mineral analysis the samples were oven-dried at 70°C for 3 days (72 h) then ground and stored in plastic vials.3·H2O), sodium acetate hydrate (C2H3NaO2·3H2O), aluminum chloride (AlCl3·6H2O), sodium hydroxide (NaOH), citric acid and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), and Rutin (C27H30O16) were purchased from Sigma-Aldrich Co. ; methanol and hexane analytical grade were obtained from HmbG Chemical Co. (Germany); Folin-Ciocalteu reagent and gallic acid were from Merck Co. . Sodium carbonate (Na2CO3) and sodium nitrite (NaNO2) were purchased from System Co. (USA). BHA (butylated hydroxyanisole), α-tocopherol (C29H50O2), hydrochloric acid (HCl), sulfuric acid (H2SO4), and hydrogen peroxide (H2O2) were purchased from Fischer Scientific Co. .2,2-Diphenyl-l-picrylhydrazyl solution (DPPH), 2,4,6-tripyridyls-triazine (TPTZ), acetate buffer, ferric chloride  and left for 2 hours in water bath shaker with 100 rpm at temperature 40 ± 1°C . The filThe TPC were determined using Folin-Ciocalteu method as reported by Singleton et al.  with som2 solution. After 6 min, 0.3 mL of a 10% AlCl3·6H2O solution was added and immediately 1.0 mL of 1 M NaOH was added. The mixture was mixed well by vortex. The absorbance was measured immediately at 510 nm with a spectrophotometer. Results were expressed as milligrams of Rutin equivalent in 1 g of sample (mg RE g−1 DW).Total flavonoid content was determined using a colorimetric method described by Abu Bakar et al. . 0.5 mL β-carotene equivalent in 1 g of sample (mg BCE g−1 DW).Total carotenoid content was determined based on the method of Khoo et al.  with sliα-tocopherol at concentrations 100 and 200 ppm were used as a standard, respectively. The absorbance of the mixture was measured at wavelength 515 nm using microplate reader . IC50 value, representing the amount of extract which scavenged/reduced 50% of the DPPH radical, was calculated from percent scavenging versus concentration curve. A higher concentration to reduce 50% of DPPH solution showed lower antioxidant activity. The calculation of % DPPH free radical scavenging is as follows:Diphenyl-2-picrylhydrazyl (DPPH) assay was used to determine the free radical scavenging activity according to the method of Brand-Williams et al.  with som3·H2O. The mixture was referred to as “FRAP reagent.” 200 μL of sample was pipetted into a test tube and mixed with 3 mL of FRAP reagent by vortexing. The mixture was allowed to react for 30 minutes at temperature of 37°C. Absorbance of the mixture was then read at 594 nm. Triplicate tubes were prepared for each extract. The FRAP values expressed in mg Trolox per g were derived from the standard curve.FRAP assay was conducted based on the method as described by Benzie and Strain  with min2SO4) was added and kept for overnight or at least 2 hours until the plant materials moistened properly. Then 2 mL of 50% hydrogen peroxide (H2O2) was added slowly and the digestion tube was placed in a digestion block where the digester block was set to heat for 45 minutes. After 45 minutes the tube was removed and allowed to cool and 2 mL of 50% H2O2 was added again, kept for heating, and cooling process was repeated until the digested solution became colorless or clear. The cleared cool sample was then filtered and the final volume was made 100 mL by adding distilled water for analysis.Plant samples were dried in an oven at 70°C for 72 h. Oven-dried samples of purslane were ground and stored in plastic vials until analysis. The N, P, K, Na, Ca, Mg, Fe, Zn, and Mn contents were analyzed using the digestion method  and deteP < 0.05).Analysis of variance (ANOVA) procedure in SAS (Version 9.2)  was usedβ-carotene equivalent (mg BCE/g dry sample)   of BHA and tocopherol standard curve. IC50 value for the 13 accessions of purslane extract in the present study was determined and varied between 2.52 ± 0.03 mg/mL and 3.29 ± 0.01 mg/mL indicating the highest antioxidant activity (2.52 mg/mL) shown by the purslane accession V12, whereas the lowest (3.29 mg/mL) was found in accession V9 among all the 13 accessions.Antioxidant activity of purslane extract that was measured by DPPH assay is presented in The ferric-reducing antioxidant power (FRAP) values were measured from all the 13 collected purslane extracts and expressed in mg Trolox per g . There wP < 0.05) in macrominerals  and microminerals  among all the 13 purslane accessions  was produced by the accessions V12, V13, V11, V11, V6, and V1, respectively, and the minimum of N, P, K, Na, Ca, and Mg  was found in accessions V4, V4, V1, V1, V10, and V2, respectively. Regarding the microelements iron (Fe) content ranged between 11.1 ± 0.18 and 1.86 ± 0.18 g/kg DW (V6 and V7), zinc (Zn) 1.48 ± 0.12 and 0.62 ± 0.06 (V12 and V9), and manganese (Mn) 1.64 ± 0.26 and 0.14 ± 0.04 g/kg DW , respectively.The correlation analysis of the selected macro- and micromineral showed that similar parameter has a highly significant correlation, while among other parameters the correlation is either nonsignificant or less significant or has moderate relation .Phenolic compounds, which are widely distributed in plants , have gaP < 0.05) difference regarding TPC, TFC, and TCC contents but Lim and Quah [Thirteen collected purslane samples were analyzed in this study for the contents of total phenolic compounds (TPC), total flavonoid contents (TFC), and total carotenoid contents (TCC). Results of the study have been presented in and Quah  reported50 is defined as the concentrations required to reduce 50% of DPPH from the original concentration. IC50 value for the 13 accessions of purslane extract in the present study was determined and the values were varied between 2.52 ± 0.03 mg/mL and 3.29 ± 0.01 mg/mL indicating the highest antioxidant activity (2.52 mg/mL) shown by the purslane accession V12, whereas the lowest (3.29 mg/mL) was found in accession V9 among all the 13 accessions. This result is partially similar to the findings of Uddin et al. [50 value) compared with both of the common purslane (V12 and V13) accessions, which represents different trends than TPC, TFC, TCC, and FRAP assays. This finding is same as the results reported by Lim and Quah [P < 0.05) difference both in % inhibition and IC50 value. It may happen due to the effect of different geographical locations and microclimatic variations as well as variations in soil qualities.In the present study antioxidant activity was analyzed utilizing 2,2-diphenyl-l-picrylhydrazyl (DPPH) free radical scavenging capacity and ferric ion reducing antioxidant power (FRAP) assay systems and all the findings are shown in n et al.  and Sirin et al.  at 60 daand Quah  who workAntioxidant activity of purslane measured by FRAP assay is presented in P < 0.05) were observed for major micro- and macronutrients among different clones collected from different locations when compared with one another both in intralocational or even in interlocational. According to the analysis results it was found that potassium (K) content was the highest among all the macro- and microminerals followed by nitrogen (N), sodium (Na), magnesium (Mg), calcium (Ca), iron (Fe), zinc (Zn), and manganese (Mn), respectively. A number of reports have indicated that purslane plants contain the maximum amount of potassium (K) [Vegetables are very rich source of essential biochemicals and nutrients such as carbohydrates, carotene, vitamins, calcium, iron, ascorbic acid, and palpable concentrations of trace minerals , 41. In sium (K) , 42, 43.Because of the reciprocal effects of Na and K authorities have argued that a high diet in potassium and low diet in sodium (low urinary Na and K ratio) favor lower blood pressure. Increase in dietary potassium as the chloride salt has shown to decrease blood pressure in some hypertensive individuals . Low Na Iron has the longest and best described history among all the micronutrients. It is a key element in the metabolism of almost all living organisms. In humans, iron is an essential component of hundreds of proteins and enzymes , 48. ManPurslane also contains trace amount of copper (Cu) is an essential trace element for humans and animals . Copper P < 0.05). K was negatively correlated with Ca and Mg, but Na showed significant negative correlation (P < 0.05) with Mg. Ca was negatively correlated with Zn and Mn, whereas Mg was also negatively correlated with Zn, Fe, and Mn. On the other hand Zn showed significant (P < 0.05) correlation with Fe, and Fe showed significant correlation with Mn at 0.05% level , higher TFC (V10), and higher TCC (V4) and antioxidant activities than the common purslane (V12 and V13). Regarding mineral constituents, the common purslane has more mineral composition than ornamental ones. So, we suggest that, both types of purslane are safe for human consumption and very good vegetable food crops for natural minerals, antioxidants, and medicinal purposes.


Azelnidipine (AZL), a long-acting dihydropyridine-based calcium antagonist, has been recently approved and used for treating ischemic heart disease and cardiac remodeling after myocardial infarction, however, its effect on hyperglycemia-induced cardiac damage has not been studied.This study examined the effect of AZL on circulating markers of cardiac damage, altered lipid and cytokines profile and markers of oxidative stress including homocysteine in diabetic rats.STZ induced diabetes caused a significant increase in blood glucose levels. It also resulted in an increase in the levels of homocysteine and cardiac damage markers, like Troponin-1, CK-MB, CK-NAC, uric acid, LDH and alkaline phosphatase. Moreover, there was an increase in the levels of proinflammatory cytokines like TNF-α, IFN-γ, and TGF-β and decrease in the levels of IL-4 and IL-10. Additionally, there was increase in the levels of cholesterol, triglycerides, LDL, VLDL and a decrease in HDL in these animals. There was an altered antioxidant enzyme profile which resulted in a notable increase in the levels of oxidative stress markers like lipid peroxides, nitric oxide and carbonylated proteins. Compared with the untreated diabetic rats, AZL treatment significantly reduced the levels of troponin-1 (P < 0.05), CK-MB (P < 0.05), CK-NAC (P < 0.05), uric acid (P < 0.05), LDH (P < 0.05) and alkaline phosphatase (P < 0.05). It also reduced the levels of the TNF-α (P < 0.05), IFN-γ (P < 0.05), and TGF-β (P < 0.05) and increased the levels of IL-4 (P < 0.05). A significant decrease in the serum cholesterol (P < 0.05), triglycerides (P < 0.05), LDL (P < 0.05), VLDL (P < 0.05) and a significant rise in levels of HDL (P < 0.05) was also observed. Treatment with AZL corrected the distorted antioxidant enzyme profile resulting in a significant decrease in the levels of lipid peroxides, nitric oxide and carbonylated proteins.Our results indicate that AZL treatment can reduce the risk of hyperglycemia induced metabolic disorders and its role can be further extended to explore its therapeutic potential in diabetic patients with cardiac complications. Improper management of metabolic disturbances especially altered lipid and protein metabolism in diabetes results in grave cardiovascular complications which causes increasing diabetes-related mortalities . Dyslipiin vivo cardiac electrical remodeling by decreasing cardiac muscle L-type calcium current and charge movement  using RA -50 semi auto analyzer by well standardized methods .Determination of lipid peroxidation  was done spectrophotometrically using the thiobarbituric acid-reactive substance (TBARS) by the method of Uchiyama and Mihara  using 1,The level of SOD and catalase enzyme activities was measured using the SOD Assay Kit-WST and Catalase Assay kit  according to the manufacturer's instructions. The concentration of reduced glutathione (GSH) was determined according to Ellman . All assNitric oxide was quantitated by using Griess Reagent Kit for Nitrite Determination  as per manufacturer's instructions.The level of protein carbonyl groups was estimated according to the method of Levine et al.  with sliSan Diego, CA, USA). A p value less than 0.05 was considered statistically significant.Data are expressed as means ± standard error (SE) and were analyzed using one-way analysis of variance and secondary analysis for significance with Tukey-Kramer post tests using Prism 4.0 GraphPad software . AZL treatment reduced the glucose levels although the levels remained elevated than the controls. AZL treatment also improved the levels of serum insulin. AZL treatment resulted in a significant decrease in the LVM/BW ratio indicating that AZL treatment prevents left ventricular hypertrophy. The data for blood glucose, serum insulin, and LVM/BW ratio has been tabulated in Table Hcy concentration in blood has been predicted as a powerful prognostic marker of cardiac complications of diabetes . STZ diaCompared to the controls, the circulating markers of cardiac damage in STZ diabetic rat, showed a significant increase in the levels of serum troponin I (1.21 ± 0.13 ng/mL vs. 0.72 ± 0.15 ng/mL), CK-MB (135.0 ± 29.5 IU/L vs. 37.50 ± 9.9 IU/L) and CK-NAC (1013.0 ± 96.3 IU/L vs. 81.21 ± 32.43 IU/L), LDH (407.0 ± 64.72 IU/L vs. 95.0 ± 10.99 IU/L) and uric acid (8.53 ± 1.54 mg/dL vs. 3.93 ± 0.5 mg/dL). This notable increase was significantly reduced by AZL treatment. The levels of troponin I (0.66 ± 0.08 ng/mL), CK-MB (38.8 ± 4.0 IU/L), and CK-NAC (150.32 ± 61.2 IU/L), LDH (61.50 ± 21.0 IU/L), Uric acid (2.1 ± 0.93 mg/dl) and alkaline phosphatase (149.6 ± 8.43 IU/L) were significantly lower when compared with the STZ diabetic group , IFN-γ (299.3 ± 42.9 pg/mL vs. 149.6 ± 17.6 pg/mL), TGF-β (2875.3 ± 349.08 pg/mL vs. 1913.3 ± 362.1 pg/mL). There was a notable decrease in levels of IL-4 (63.8 ± 11.1 pg/mL vs. 105.9 ± 24.2 pg/mL) and IL-10 (462.4 ± 31.2 pg/mL vs. 979.5 ± 150.3 pg/mL). AZL treatment prevented the increase in the levels of TNF-α (90.0 ± 14.8 pg/mL), IFN-γ (200.2 ± 38.1 pg/mL), and TGF-β (1892.4 ± 321.0 pg/mL). It also resulted in an increase in the levels of IL-4 (132.1 ± 22.9 pg/mL), which was even higher than the controls , AST (86.7 ± 10.5) and alkaline phosphatase (330.0 ± 12.51 IU/L) compared with those of the controls viz. 37.5 ± 11.5, 27.7 ± 2.5 and 181.7 ± 32.9 IU/L, respectively. After treatment with AZL, the levels of ALT, AST and alkaline phosphatase were observed to be normalized at 27.5 ± 5.2 for ALT, 32.0 ± 4.0 for AST and 149.6 ± 8.4 IU/L for alkaline phosphatase, indicating that AZL treatment does not metabolically induce xenobiotic hepatotoxicity . AZL treatment significantly reduced the levels of lipid peroxidation  which was very close to the control group  and AZL treatment significantly prevented this increase  . This nitric oxide level after AZL treatment were reduced  and were close to control group  which is a direct marker for oxidative stress increased significantly in the cardiac tissue of STZ group compared with the control  Figure . Levels p Figure .P < 0.05 and GSH 57.9 ± 32.3 vs. control 493.5 ± 46.2, P < 0.05), whereas level of catalase (mU/mg protein) increased significantly compared with controls . These different enzyme and non-enzyme antioxidant levels were normalized after AZL treatment   and GSH (ng/mg protein) were depressed significantly in the diabetic rat  Figure .Diabetes is a metabolic syndrome with a cluster of common clinical disorders that is related with an increased risk for cardiovascular disease . DespiteDiabetic cardiomyopathy characterized by diastolic dysfunction and left ventricular hypertrophy is usually the terminal condition of heart in diabetes . Our resElevated hcy level is also an independent risk factor for the development of atherosclerosis as well as a prognostic marker in ischemic heart disease ,54. ProtDiabetes is associated with an increase in the oxidative stress that results from exhaustion of cellular antioxidants and depletion of antioxidant enzymes . The resIt is widely accepted that high levels of circulating cardiac damage markers like troponin I, CK-MB, CK-NAC, LDH and uric acid represent a powerful and sensitive predictor of increased cardiac complications . In thisApart from the altered metabolic condition, diabetes is also an inflammation-prone condition. Hyperglycemia-induced ROS stimulates signal transduction to instigate inflammatory cytokines, e.g. TNF-α, IFN-γ and TGF-β . This leDiabetes Care27Full Text.In our study, the altered lipid profile observed in the diabetic condition was also abrogated by AZL treatment. AZL improved the serum cholesterol, triglycerides, LDL and VLDL. It also improved the markers of atherogenic risk, . These findings suggest that AZL possesses lipid lowering activities due to its unique anti-atherogenic properties which also contribute additionally to its cardiovascular protective actions. This is the first report which evaluated the positive effect of AZL on lipid profile in STZ-diabetic rats In conclusion, we showed that AZL treatment abrogated the metabolic disorders in STZ-diabetic rats at least partly through its antioxidant and antiatherogenic properties. These results indicate that the protective effect of AZL might be, at least in part, due to its inhibitory ability on free radical formation, or due to its free radical scavenging ability. These data also indicate that AZL exerts cardioprotection due to its unique modulatory and antioxidative property and thus may be a promising 'cardioprotective DHP' that can even target the secondary cardiac complication of diabetes. This property of AZL could be further extended to clinics and can be used as adjuvant therapy in cardiac complications especially in diabetes.The authors declare that they have no competing interests.VK and SK contributed equally to the experimental designing and bench work. ASP performed the Hcy estimation and SLS conceived and designed the study. All authors have read and approved the final manuscript.


This study analyzes in vitro reduction of hexavalent chromium using cell free extract(s) of the fungus that was characterized based on optimal temperature, pH, use of electron donors, metal ions and initial Cr(VI) concentration in the reaction mixture. This showed the highest activity at 37°C and pH 7.0; there is an increase in Cr(VI) reductase activity with addition of NADH as an electron donor, and it was highly inhibited by Hg This metal is used in the tanning of hides and leather, the manufacture of stainless steel, electroplating, and textile dyeing and used as a biocide in the cooling waters of nuclear power plants, resulting in chromium discharges causing environmental concerns . Cr exissolution . These tsolution , and . The objective of this study was to analyze in vitro reduction of Cr(VI) by cell free extracts of 2PO4, 0.20% MgSO4, 0.50% (NH4)2SO4, 0.50% NaCl, and 0.25% glucose) supplemented with 500 mg/L K2CrO4; the pH of the medium was adjusted and maintained at 5.3 with 100 mMol/L citrate-phosphate buffer. The cultures were incubated at 28°C for 7 days. The strain was identified based on its morphological structures such as the color, diameter of the mycelia, and microscopic observation of formation of spores [A chromate-resistant filamentous fungus was isolated from polluted air with industrial vapors, near the Chemical Science Faculty, located in the city of San Luis Potosí, Mexico, in Petri dishes containing modified Lee's minimal medium   and harvested by centrifugation at 3000 ×g at 4°C; cell pellets (10 mL) obtained on centrifugation were washed twice with 100 mM potassium phosphate buffer (pH 7.0) and resuspended in the same buffer. Triplicates of these suspended cell pellets were spiked with Cr(VI) concentrations of 2–10 mg/100 mL, vortexed for 30 min, and incubated at 30°C for 6 h. Heat-killed (2 mL) culture pellets were used as control. After 6 h incubation, the tubes were centrifuged, and 100 µL aliquots were withdrawn from each sample to estimate the remaining Cr(VI) by 1,5-diphenyl carbazide (DPC) method [Culture suspensions of ) method .Penicillium sp. was grown for 4 days, harvested, and washed with potassium phosphate buffer (pH 7.0) as described above. The suspended culture pellets were treated with 0.2% (w/v) sodium dodecyl sulphate, 0.2% tween 80, (v/v), 0.2% Triton X-100 (v/v), and 0.2% toluene (v/v), by vortexing for 30 min to achieve cell permeabilization. Permeabilized cell suspensions (0.5 mL) were then added with 2–10 mg/100 mL of Cr(VI) as final concentrations and incubated for 6 h at 30°C. Experiments with each set of permeabilization treatment and Cr(VI) concentrations were performed in triplicates.Bacterial culture of Penicillium sp. were prepared by modifying the previously published protocols [Cell-free extracts (CFE) of rotocols . Fungal µL of 100 mM potassium phosphate buffer (pH 7.0) added with 250 µL aliquots of CFE for chromate reduction and 50 µL of NADH. The system volume of 1.0 mL was kept constant for all experiments. Enzymatic chromate reduction was estimated as described previously using a standard curve of Cr(VI) 0–30 mM . Assay m+, Ca2+, Cu2+, Hg2+, Mg2+, Cd2+, and Fe3+ were tested by using 10 mM solutions of Na2SO4, CaCl2, CuCl2, HgCl2, MgCl2, CdCl2, and FeCl3. The electron donors tested were NADH, glucose, sodium acetate, formic acid, citrate, cystin, lactic acid, and ascorbic acid in a final concentration of 1 mM, and the inhibitors were EDTA, KCN, NaN3, and β-mercaptoethanol at the same concentration. Unit enzyme activity for chromate reductase was derived as amount of enzyme that reduces 1 mMol of Cr(VI) per min at 37°C. Specific activity was defined as unit chromate reductase activity per minute per mg protein concentration in the CFE. Protein concentrations were estimated using Lowry method [Chromate reductase activity was measured at 37°C at different pH values using several buffers . The effect of temperature was studied by measuring chromate reductase activity at different incubation temperatures between 20 and 60°C, at pH optimum. The CFE samples were also treated with several metal ions to a final concentration of 1 mM at optimal pH and temperature; Nay method . A. niger and A. parasiticus [Fusarium solani [Paecilomyces lilacinus [Pseudomonas sp. [The resting cells of the fungus were expedient in reducing 0–10 mg/100 mL Cr(VI) concentrations in 8 hours as shown in asiticus  Fusariumm solani , Paeciloilacinus , and theonas sp. . Structuonas sp. .µmoles/min/mg protein. These results indicate that the Cr(VI) reductase was associated with the CFE. Fungal, yeast, and bacteria chromate reductases have been localized either in CFE of A. niger and A. parasiticus [Pichia jadinii M9, and Pichia anomala M10 [ Pichia sp. [ and Bacillus sp. [C. maltosa [Pichia sp. [Pannonibacter phragmitetus [Pseudomonas sp. G1DM21 [ Bacillus megaterium [Enterobacter cloacae [The cell permeabilization increased the Cr(VI) reduction by the resting cells, as the permeabilized cells with Triton X-100 which could reduce 57%, Toluene 52%, SDS 47.4%, and Tween 80, 40.4%  of 30 mMasiticus , Pichia mala M10 , Pichia chia sp. , and Bacllus sp. , and cyt maltosa , Pichia chia sp. , and Pangmitetus  and memb. G1DM21 , Bacillugaterium , and EntPenicillium sp. was characterized in different in vitro conditions. To define the optimal pH, the Cr(VI) reductase assays were carried in potassium phosphate, citrate phosphate, and Tris-HCl buffers of differential pH ranges; of the different buffers used the potassium phosphate buffer showed a characteristic pH curve for the enzymatic activity with an optimum pH of 7.0, as depicted from A. niger and A. parasiticus [P. jadini M9 [Pseudomonas sp. G1DM21 [E. coli CFE [Bacillus sp. [The functioning of the chromate reductase of asiticus  and the adini M9 . Other a. G1DM21 , 6.5 andcoli CFE , and in llus sp. .P. jadinii M9, incubation at 55°C produced a reduction in activity of 55% [P. anomala when incubated at 8°C, a decrease in activity of 25% was observed, and at 50°C the activity was 50%. For A. niger and A. parasiticus [Pseudomonas sp. G1DM21 [E. coli a [Bacillus sp. CFEs [Pseudomonas putida CFE probed to be more resistant, keeping its stability up to 50°C [The optimal temperature for the Cr(VI) reductase activity was 37°C, but the reductase activity was altered significantly at 20°C (39% of inhibition), but when the assays were performed at 50°C temperature the reductase activity we found 14.2% of inhibition asiticus , Pseudom. G1DM21 , E. coli. coli a , and Bacsp. CFEs , the thesp. CFEs , 42. On  to 50°C .Penicillium sp. was determined as exhibited in 2+ and 40.2% with Na+, and these results are different than those reported by the yeast P. jadinii M9 Chromate reductase because only Cu2+ and Na+ produced an augmentation in the activity of 63 and 30%, respectively [2+, while addition of Mg2+, Fe3+, Ca2+, and Cd2+ resulted in a decrease of activity between 40% and 51%. In P. anomala M10 chromate reductase, only Cu2+ produced a raise in activity of a 31%. Inhibition by Hg2+ was higher in P. anomala and Pseudomonas sp. than in Penicillium sp. with a decrease in activity of 85% and 90%, respectively [2+ and Mg2+ was approximately 50%, while Fe3+ reduced the activity by 32%. These results agree with those reported for Arthrobacter crystallopoietes [Bacillus sp. [2+ can be related to its affinity to –SH ligands, then suspecting the presence of this chemical group in the active site of the enzyme related to chromate reductase activity [The effect of different metal cations on the chromate reductase activity of ectively , and allectively , 37. Inhopoietes , and Bacllus sp. . On the activity .P. jadinii M9 and P. anomala Chromate reductase with NADH [Pseudomonas sp. with citrate, acetate, glucose, and formate [Bacillus sp., glucose has been reported to act as an electron donor and has been demonstrated to increase Cr(VI) reduction [Shewanella putrefaciens MR-1 [The reductase activity increased on supplementation in the reaction mixtures with electron donors. All the electron donors analyzed increased the activity, and the most efficient were ascorbic acid, NADH, glucose, and citrate by 4.4, 4.0, 2.9, and 2.87 times, respectively , and theith NADH  and Pseu formate . In preveduction , 46, andens MR-1 . Our worens MR-1 , 42, 43.ens MR-1 , the oxiE. coli ATCC 33456 19, [Bacillus subtilis [S. putrefaciens MR-1 [Bacillus sp. ES29 [ Respiratory inhibitors like azide (1 mM), EDTA (1 mM), and cyanide (1 mM) caused inhibitions of 48%, 47%, and 32%, respectively , in the 3456 19,  and aerosubtilis  and inhiens MR-1  while nosp. ES29 . Respirasp. ES29 . Penicillium sp. Chromate reductase assays of the cell-free extracts (CFE) have shown a high Cr(VI) reductase activity. The Cr(VI) reduction potential of the resting cells was increased by cell permeabilization. Optimum temperature and pH of chromate reductase activity of the bacterium were found to be 37°C and 7.0, respectively, and activity was enhanced in presence of 0.1 mM NADH and other electron donors. 1 mMol of metal ions like Cu2+, Na+, Hg2+, Mg2+, Fe3+, Ca2+, and Cd2+ and respiratory inhibitors resulted in a decrease of the activity. Finally, the results of higher rates of Cr(VI) reduction by the CFE, functionality of the Cr(VI), indicate a potential application of Penicillium sp. for Cr(VI) bioremediation.The present study analyzed a very efficient Cr(VI) reductase of


Cell lines were exposed to compound for 3–6 days and primary cells for 4 days. The viability of primary cells was additionally evaluated 3, 7, and 31 days after removal of tested compound to determine the durability of the response. Our studies indicate that CNDAC and mitoxantrone have a greater impact on viability than ara-C in primary AML cells and AML cell lines. CNDAC is more effective at reducing viability and inducing apoptosis than ara-C at equivalent concentrations in the THP-1 cell line, which is defined as displaying resistance to ara-C. As sapacitabine has shown in vivo activity at clinically achievable doses, future studies are warranted to assess the potential for combining it with ara-C and/or mitoxantrone, with an emphasis on cells and patients insensitive to ara-C treatment.Achieving improvements in survival and reducing relapse remains a challenge in acute myelogenous leukemia (AML) patients. This study evaluated the  Acute myeloid leukemia (AML) therapy is continually challenged by high incidences of disease relapse and patient mortality. The overall 5-year survival rate for AML is 30–40% for patients >45 years and <10% for patients over 60 years , 3. The Nucleoside analogs, such as ara-C, represent a major group of antileukemic agents . They arβ-d-arabino-pentofuranosylcytosine (CNDAC), the major metabolite of oral drug sapacitabine, is a nucleoside analog that is structurally related to ara-C and gemcitabine [in vitro efficacy of CNDAC, this study compared CNDAC to conventional drugs, ara-C and mitoxantrone, in the promyelocytic cell line HL-60, which is known to be sensitive to ara-C, the monocytic cell line THP-1, known to be less sensitive to ara-C, and peripheral blood and bone marrow cells from 5 AML patients. Mitoxantrone is a synthetic anthracenedione that was developed as an analog to doxorubicin to reduce drug associated cardio-toxicity . It is wcitabine , 14. Thecitabine . Unlike citabine , 16. Thicitabine . Replicaμg/mL streptomycin solution (Thermo Scientific Hyclone), and 2 mM L-Glutamine (Thermo Scientific Hyclone). Cells were cultured at 37°C, 5% CO2, and 100% humidity. Cells were in the logarithmic growth phase at the beginning of all experiments. AML cell lines, HL-60 and THP-1, were obtained from ATCC  and cultured in RPMI 1640 medium (with phenol red)  supplemented with 10% fetal bovine serum (Thermo Scientific Hyclone), 100 U/mL penicillin/100 4 cells/well. Wells were gelatinized by adding 0.1% gelatin in water  to each well and incubating for 6 hours. The M2-10B4 stromal layer was incubated at 37°C, 5% CO2, and 100% humidity for at least 24 hours before addition of AML cells.M2-10B4 stromal cells were purchased from ATCC and cultured in RPMI 1640 media. Stromal layers were prepared by irradiating cells at 80 Gy and plating them on gelatinized 24 well plates at a concentration of 5 × 10μL with 56.6 to 98.5% blasts in the bone marrow. Additional clinical information is provided in μg/mL streptomycin, 2 mM L-Glutamine, 100 ng/mL SCF , and 50 ng/mL IL-3 . Co-culture media was the same as suspension medium with the addition of 1 μM hydrocortisone .Peripheral blood (PB) and bone marrow (BM) specimens were obtained from 5 AML patients with informed consent on an IRB approved protocol in accordance with the Declaration of Helsinki. Of these patients, 3 were newly diagnosed and 2 had relapsed. The 2 relapsed patients had trisomy 13 and complex karyotypes, respectively. All other patients had normal cytogenetics. Total WBC counts of the patients ranged from 27.06 to 93.75 K/Cytarabine (ara-C) was purchased from Bedford Laboratories  and mitoxantrone from Mayne Pharma Limited . CNDAC was kindly provided by Cyclacel Ltd. . Stock concentrations for ara-C (100 mM) and mitoxantrone (1 mM) were made in Dulbecco's Phosphate-Buffered Saline (DPBS) and stored at −80°C while CNDAC (100 mM) was dissolved in dimethylsulphoxide (DMSO) (Sigma Aldrich) and stored at −20°C. Working stocks of drugs were made in media.6 cells/mL (low) or 0.5 × 106 cells/mL (high). Cells were treated with ara-C or CNDAC at 0.5, 1, 2, 3, 4, 5, and 10 μM and mitoxantrone at 0.0025, 0.005, 0.01, 0.02, 0.03, 0.04, and 0.05 μM in triplicate at 37°C, 5% CO2, and 100% humidity. Appropriate untreated controls were included. Cells were analyzed 3, 4, 5, and 6 days posttreatment. Cell lines (HL-60 and THP-1) were treated in suspension in 48 well plates at seeding densities of 0.05 × 106 primary BM and PB cells were treated with 1 μM (low), 10 μM (medium), and 100 μM (high) of ara-C or CNDAC or 0.005 μM (low), 0.05 μM (medium) and 0.5 μM (high) mitoxantrone in 24 well plates at 37°C, 5% CO2, and 100% humidity for 4 days. Appropriate untreated controls were included. Postdrug treatment, both PB and BM non-adherent cells were washed to remove compound, replated on M2-10B4 stromal layers, and reincubated at 37°C, 5% CO2, 100% humidity. Cells were analyzed immediately posttreatment and following 3, 7, and 31 days postdrug removal.1 × 1050 values in AML cell lines. HL-60 and THP-1 cells were plated on 96 well flat bottom plates at 5 × 103 cells/well. Cells were incubated at 37°C, 5% CO2, 100% humidity for 24 hours before addition of drugs. Cells were treated with ara-C, CNDAC and mitoxantrone in triplicate at 10 concentrations between 0.005 and 100 μM. Plates were reincubated for 72 hours after addition of drugs. After 72 hours, Alamar Blue  was added to all wells at a final concentration of 10%, and plates were returned to the incubator for 8 hours before absorbance was read on the spectrometer  at wavelengths of 570 nm and 600 nm. Cell proliferation was determined by calculating the reduction of Alamar Blue. The equation used was as follows:The Alamar Blue assay was performed to determine drug ICO and R are the molar extinction coefficients of Alamar Blue in its oxidized and reduced form, where O600 = 11726, O570 = 80586, R570 = 155677 and R600 = 14652. N is the absorbance of the negative control well. % Inhibition was calculated using the equation:At each time point, cell lines and primary AML cells were assessed for overall viability by obtaining counts on a hemocytometer slide using trypan blue exclusion dye. Percentage live and dead cells in cell lines were calculated from the raw counts. For primary cells, Percentage of survival was calculated by dividing remaining live cells by the initial cell number.−AnnexinV−) were considered live and non-apoptotic/healthy. Cells that stained positive only for AnnexinV (7AAD−AnnexinV+) were early apoptotic and those that stained positive for both 7-AAD and AnnexinV (7AAD+AnnexinV+) were late apoptotic or necrotic. The sum of cells in the 7-AAD−AnnexinV+ and 7-AAD+AnnexinV+ quadrants were total dead cells.Cells were washed twice in cold DPBS and resuspended in binding buffer containing appropriate volumes of 7AAD and AnnexinV and incubated in the dark for 15 minutes at room temperature. Excess antibody/dye was washed off, and data was acquired on the flow cytometer within 1 hour of staining. Cells were evaluated on a plot of 7-AAD versus AnnexinV. Cells that stained negative for 7-AAD and AnnexinV .Data are expressed as mean values ± SEM. Comparisons between drug treatments in cell lines were performed using the Students 50 values, defined as the half-maximal inhibitory concentration, for ara-C and CNDAC in HL-60 and THP-1 cells obtained by Alamar Blue assay  with the % cell death ranging from 6.26 ± 1.39 to 63.8 ± 5.35 on day 3 (n = 3). However, ara-C induced higher cell death in cells plated at the lower density with 16.2 ± 7.33 to 94.0 ± 2.82 % cell death on day 3  . However, at lower seeding densities, equivalent concentrations of CNDAC were more effective than ara-C in inducing cell death  . At bothll death . The IC5e points .μM was 12.1 ± 0.28 on day 3 (n = 3). THP-1 cells had an overall low response to CNDAC on day 3, however, cell death was significantly higher than ara-C at doses >2 μM . There was also a significant increase in cell death for cells treated with ≥2 μM CNDAC on day 4 as compared to day 3 . The % of cell death for CNDAC-treated cells ranged from 39.9 ± 4.08 (2 μM) to 54.6 ± 3.08 (10 μM) on day 4 and 49.8 ± 1.55 to 86.9 ± 2.17 on day 6 (n = 3). Comparatively, at the lower seeding density, ara-C induced cell death at drug concentrations between 0.5 μM and 10 μM was highest on day 3—1.52 ± 1.67 % to 55.6 ± 9.87%   . IC50 va density . μM) to 90.8 ± 2.72 (0.05 μM) on day 3 (μM) as compared to the untreated for HL-60 but not THP-1 cells . At the lower seeding density, mitoxantrone was able to induce ≥84.9 ± 4.76% cell death in HL-60 cells on day 3 and only ≥30.7 ± 11.1% in THP-1 cells at doses of 0.0025 μM and above , the percent survival of PB and BM cells was 125.91 ± 38.7% and 130.91 ± 35.8% respectively. There was no further cell expansion beyond day 7 as survival was 130.83 ± 68.3% for PB and 129.24 ± 55.5% for BM cells on day 35 (31 days after replating).Suspension and coculture systems for primary AML PB and BM MNCs were used during the first 4 days in order to mimic μM) treated PB cells. However, 10 μM ara-C induced a significant reduction in cell survival on days 4 and 7 when compared to the untreated . Although not significant, the survival of PB cells trended lower than untreated  at 4 days post low-dose CNDAC treatment. By day 7, however, survival of CNDAC-treated cells at this dose was significantly lower than the untreated . Treatment with 10 μM (medium dose) CNDAC resulted in a significant drop in cell survival compared to the untreated on days 4, 7, and 14 . On day 35, cell survival still trended lower, but was not significant  (μM) mitoxantrone-treated cells had significantly lower cell survival on days 4 and 7  when compared to the untreated. Treatment with 0.05 μM (medium dose) of mitoxantrone led to a significant loss in cell survival on all analysis days  . At the high dose (100 or 0.5 μM), all three drugs induced significant loss of survival of PB cells as compared to the untreated    . Low dos, n = 5) . The ove, n = 5) .P = 0.056; n = 5). Unlike PB, the response of BM cells to 10 μM ara-C was not statistically different from the untreated controls on days 4 and 7 . The profile of medium-dose CNDAC (10 μM) and mitoxantrone (0.05 μM) treated cells when compared to untreated controls was similar to that seen in the PB   .μM CNDAC or 0.005 μM mitoxantrone as compared to 1 μM ara-C   . A simil, n = 5) . Individ50's of the drugs. Conversely, at high seeding densities, cells have lower proliferation rates thus requiring higher doses of drug to achieve similar effects (μM), indicating that higher doses are unnecessary. A low effective dose is highly desired in the clinical setting, as low doses usually equate to less toxicity and meylosuppression in patients.This study compared the cytotoxic effects of novel agent CNDAC to conventional agents, ara-C and mitoxantrone. The activity of ara-C and CNDAC is cell cycle dependent. Cell lines plated at low seeding densities have higher proliferation rates indicative of actively dividing cells thereby lowering IC effects . Regardl50 = 7.7 μM). Higher cell death for ara-C-treated THP-1 cells on day 3 compared to day 6, when plated at the low density, can be explained by increasing amounts of CDA in the culture media resulting from active proliferation of cells. At the high seeding density, an IC50 for the drug in THP-1 cells was not achievable at the doses tested, perhaps due to complete inactivation of ara-C by CDA. THP-1 cells, however, responded to CNDAC with IC50 values ≤2.774 μM, regardless of cell density  activity, which deaminates ara-C into the inactive ara-U , 8, 18.  density . The IC5ll lines  and micell lines . Also, Pines [μM  thus expines [μM . This isStromal cells are often thought of as sanctuaries for primary leukemic cells. They produce cytokines and growth factors that protect cells in their niche and influence their proliferation, differentiation, and survival –26. The In our experiments, while PB cells seemed more sensitive to effects of drugs, a fraction of BM cells was protected from toxic effects as they were treated with drug in the presence of stroma. At the low and middle doses, both PB and BM cells treated with ara-C seemed to grow out post drug removal with the support of the stromal cells . Howeverβ elimination intermediate of CNDAC, is a poor substrate for DNA chain elongation and is responsible for inducing DNA single strand breaks. It is known to have a long shelf life in whole cells and its removal by the nucleotide repair (NER) mechanism is thought to be a slow process [Unlike Ara-C treated cells, the residual cells, in both BM and PB after CNDAC-treatment, did not expand in culture. 2′- C-cyano- 2′, 3′-didehydro-2′,3′ -dideoxycytidine (CNddC), the chain terminating  process . This unin vivo in a P388 mouse leukemia model [Our experiments have shown CNDAC to be more active than ara-C in both primary cells and cell lines. Sapacitabine is the palmitoyl derivative of CNDAC. The palmitoyl chain allows for oral absorption of the drug and protects the N4 amino group from deamination. A comparison of CNDAC and sapacitabine is presented by Serova et al.  The dataia model , 33. ActOn the clinical front, sapacitabine has shown promising antileukemic activity. Phase I trials demonstrated the drug to be safe and active in the treatment of certain hematologic disorders (MDS and AML) and solid tumors alike . It is aFuture studies are warranted to assess the potential of combining CNDAC in the upfront setting of AML either simultaneously or in sequence with standard chemotherapy; particularly in patients who do not respond to ara-C-based therapies. There is a lifetime limit for the administration of mitoxantrone to patients as its toxicity is accumulative . CNDAC,


In conclusion, we show that fluoxetine exerts both harmful and beneficial age-dependent effects on depressive behavior, body weight and wakefulness, which may relate, in part, to differential fluoxetine-induced neuroplasticity in the amygdala.The selective serotonin reuptake inhibitor (SSRI) Prozac® (fluoxetine) is the only registered antidepressant to treat depression in children and adolescents. Yet, while the safety of SSRIs has been well established in adults, serotonin exerts neurotrophic actions in the developing brain and thereby may have harmful effects in adolescents. Here we treated adolescent and adult rats chronically with fluoxetine (12 mg/kg) at postnatal day (PND) 25 to 46 and from PND 67 to 88, respectively, and tested the animals 7–14 days after the last injection when (nor)fluoxetine in blood plasma had been washed out, as determined by HPLC. Plasma (nor)fluoxetine levels were also measured 5 hrs after the last fluoxetine injection, and matched clinical levels. Adolescent rats displayed increased behavioral despair in the forced swim test, which was not seen in adult fluoxetine treated rats. In addition, beneficial effects of fluoxetine on wakefulness as measured by electroencephalography in adults was not seen in adolescent rats, and age-dependent effects on the acoustic startle response and prepulse inhibition were observed. On the other hand, adolescent rats showed resilience to the anorexic effects of fluoxetine. Exploratory behavior in the open field test was not affected by fluoxetine treatment, but anxiety levels in the elevated plus maze test were increased in both adolescent and adult fluoxetine treated rats. Finally, in the amygdala, but not the dorsal raphe nucleus and medial prefrontal cortex, the number of PSA-NCAM (marker for synaptic remodeling) immunoreactive neurons was increased in adolescent rats, and decreased in adult rats, as a consequence of chronic fluoxetine treatment. No fluoxetine-induced changes in 5-HT However, in 2006 fluoxetine was approved in children aged 8 years and older for treatment of moderate to severe depression Selective serotonin reuptake inhibitors (SSRIs) are among the most widely prescribed drugs in psychiatry. While numerous trials have shown robust safety of SSRIs in adults, limited data are available on their short- and long-term safety in adolescents. Yet, the number of children for whom anti-depressants are prescribed has increased during the last decade th week of age had no effect on depression-like behavior in mice. Also chronic adolescent fluoxetine treatment (10–18 mg/kg) between 3 and 7 weeks of age did not affect adult measures of anxiety-, fear- or stress-related behaviors in mice Some recent rodent studies have elaborated the human findings. Mason et al. The present study aimed to increase the understanding of the age-related outcomes of adolescent fluoxetine exposure. Since the mixed results reviewed above may be due to differential wash-out periods and ages of testing, and the adverse effects of adolescent fluoxetine exposure in humans are manifested particularly short after the start of treatment 1A receptor and PSA-NCAM in the dorsal raphe nucleus (origin of serotonergic cell bodies), amygdala, and medial prefrontal cortex (mPFC), brain areas playing a central role in emotional, cognitive and sensory information processing and responsive to the (therapeutic) effects of antidepressants 1A receptor is strongly implicated in the actions of SSRIs 1A receptor, immunoreactivity was differentially affected by fluoxetine in adolescent and adult rats.There is accumulating evidence that SSRIs exert their effects through neuroplastic changes  male rats  were 21 and 63 days of age at arrival. Since they were obtained from the same experimental animal supplier and both age groups arrived at the animal facility 4 days before the start of the treatment, the background and history of both age groups was similar. The animals were housed two per cage (Macrolon® 40×25×15 cm) in temperature controlled rooms (20±2°C). After 4 days of acclimatisation, the rats were daily treated with 12 mg/kg fluoxetine  or 1% methylcellulose  by oral gavage for 21 days. Rats treated during PND 25–49 are referred to as the adolescent group, and rats treated during PND 67–88 represent the adult group. Body weight was monitored daily throughout the treatment. From 7 days after the last injection the animals were tested as described below. Separate groups of animals were used for 1) the open field and elevated plus maze tests , 2) the acoustic startle/prepulse inhibition and forced swim tests , and immunohistochemistry , and 3) sleep-wake behavior and plasma (nor)fluoxetine levels . Consecutive tests were separated by 2 days , with a standard cortical tripolar electroencephalography (EEG) electrode set  and a bipolar electromyography (EMG) electrode set (Plastics One MS 303/71). EEG electrodes were placed in the frontal cortex and in the parietal region, with coordinates A 2.0, L 3.5 and A −6.0, L 4.0, respectively . The cages (45×45×45 cm), made of transparent Perspex walls and a black floor, were equipped with a feeding station and two drinking bottles. Each cage had a top unit containing a built-in digital infrared-sensitive video camera, infrared lighting sources, and hardware needed for video tracking. The rats were placed in the Phenotyper cages ©, Nijmegen, The Netherlands). Results were expressed as the mean of time spent (s) in open arms.The apparatus, made of polyvinylchloride, was elevated to a height of 50 cm with two open  and two enclosed  arms. As described earlier Cylindrical glass tanks , filled to a depth of 30 cm with 22 (+/−1)°C water, were used. After a 15-min water experience on day 1, the animals were tested 24 hrs later in the water cylinders for 5 min The acoustic startle chambers consisted of a Plexiglas tube  with a piezoelectric accelerometer mounted beneath the tube. The acoustic stimuli were delivered by the PSR2 computer software, via a speaker that was placed 10 cm above the tube. The software converted the accelerometer measurements into a digital signal. The background noise was 70 dB. Each session started with 5 min acclimatisation, followed by ten blocks of 5 trials consisting of one 120 dB startle stimulus , a no stimulus condition, and pre-pulse startle stimuli of 3, 5 or 10 dB above background (delivered pseudo-randomly). The prepulses were always followed by the 120 dB stimulus after 100 ms. All stimuli were delivered for 20 ms. The interval between each trial was 10/20 s. The startle amplitude was calculated as an average of the 10 trials of the startle trial and the three different prepulse trials. The % PPI was calculated as follows: 100 -  ×100.Rats were kept in a Perspex recording cage (30×25×35 cm) equipped with a passive infrared movement detector  attached to the ceiling of the cage. Rats, chronically provided with cortical EEG electrodes and nuchal EMG electrodes, were connected to a swivel, which allowed free movement in their recording cage. EEG and EMG signals were amplified, band-pass filtered , a notch filter eliminated 50 Hz, sampled at 256 Hz and stored on disk with the aid of a WINDAQ data acquisition system . We analyzed 4 hrs  from each rat, using the WinDaq Waveform Browser . Sleep and wake states were visually scored according to conventional criteria based on EEG and EMG 5 hrs and 1 wk after the last fluoxetine injection blood samples were collected in Microvette CB 300  tubes through a tail cut. Blood plasma was obtained by centrifugation of the blood at 4°C at 4000 rpm for 15 min. Supernatant was stored at −20°C until use.50–100 µl of the plasma samples, containing fluvoxamine as internal standard, were extracted as described by Duverneuil et al. Norfluoxetine and fluoxetine were purchased from Sigma, HPLC grade acetonitril and hexane from Biosolve B.V. , isoamylalcohol and hydrochloric acid from Merck , and sodiumhydroxide, phosphoric acid and citric acid monohydrate were obtained from Acros .1A , or PSA-NCAM  antisera.7 Days following the forced swim test, rats of group 2 were deeply anesthetized and perfused transcardially with 0.1 M PBS, pH 7.3, followed by 400 ml 4% paraformaldehyde dissolved in 0.1 M PB, pH 7.2. Immunostaining was performed as previously described 1A immunoreactivity was quantified using Image J, a public domain image processing program (http://rsb.info.nih.gov/ij/) that assessed the intensity of immunostaining Numbers of PSA-NCAM immunopositive cells were quantified post-hoc testing (time effect sleep/wake behavior), two-way ANOVA  or repeated measures ANOVA . Interaction effects were further analysed using two-way ANOVA (sleep/wake patterns) and Student t-tests. Probability values of p<0.05 were considered significant. NS  =  not significant.All statistical analyses were performed using SPSS version 16.0 . Data were analyzed using one-way ANOVA followed by Bonferroni  = 0.6, NS) and adult  = 0.474, NS) fluoxetine and methylcellulose groups. Repeated measures ANOVA revealed that fluoxetine had no effect on body weight in adolescent rats  = 0.826, NS), but significantly reduced adult body weight  = 9.218, p<0.01). Independent Student t-tests indicated that the body weight reduction in adults was significant (p<0.05) from day 11 of treatment and further on. Similar results were obtained for group 2 and 3 (data not shown).The bodyweight of fluoxetine and methylcellulose treated animals (group 1) across the fluoxetine/methylcellulose treatment is shown in  = 5.073, p<0.05). However, fluoxetine did not affect locomotor activity  = 0.687, NS)  = 1.662, NS).Two-way ANOVA revealed that total distance moved was significantly higher in adolescent compared to adult rats  , and no  = 9.344, p<0.005), but there was no significant age  = 0.803, NS) nor age x treatment  = 0.17, NS) effect (Fluoxetine significantly decreased open arm time in adolescent and adult rats (F) effect . = 12.544, p<0.001). Further, we obtained a significant age x treatment interaction  = 5.467, p<0.05), but no significant treatment effect was observed  = 0.004, NS)  = 2.107, p<0.05), but had no effect in adults  = 1.385, NS).Adolescent rats spent less time on floating (immobility) compared to adult rats  . A subse = 49.006, p<0.0001), there was a significant treatment  = 4.570, p<0.05), and age x treatment  = 4.465, p<0.05) effect  = 2.138, p<0.05) but not in adolescents  = 0.731, NS).Overall, the adult animals showed a higher startle reflex compared to the adolescent rats (F) effect . A subse = 5.755, p<0.05), but no significant treatment  = 2.395, NS) or age x treatment  = 1.956, NS) effect was observed  = 14.691, p<0.0001), treatment:  = 0.287, NS), age x treatment:  = 0.711, NS)] and PP10 .When the animals were exposed to prepulse stimuli of 73 (PP3), 75 (PP5) or 80 (PP10) dB preceding the startling stimulus, four animals had to be removed from the analysis because PPI scores were negative. At PP3, PPI was higher in adult compared to adolescent rats  = 1.900, NS), treatment  = 0.168, NS) and age x treatment interaction  = 0.337, NS) effects, but the time x age  = 5.920, p<0.001) and time x age x treatment interactions  = 3.146, p<0.05) were significant. Posthoc analysis of the time x age x treatment interaction indicated that there were age x treatment interactions within the 17.30–18.30  = 15.173, p<0.05) and 19.30–20.30  = 5.745, p<0.05) intervals. The treatment effects in the 17.30–18.30 interval was significant for the adult  = 2.809, p<0.05), but not adolescent  = 0.346, NS) rats. Thus, fluoxetine increased the awake state in adult rats only. For the 19.30–20.30 interval Student's t-test did not reveal treatment effects for either the adult or adolescent rats (t<1.9). An age effect was obtained for the 17.30–18.30 interval  = 17.613, p<0.05), reflecting increased time spent in the awake state in adolescent rats. Finally, one-way ANOVA (using time as between-subject factor) revealed a significant effect of time  = 20.482, p<0.0001). According to a subsequent Bonferroni post-hoc test time in the awake state was significantly higher in the 17.30–18.30 interval compared to the other intervals (p<0.005), and significantly lower in the 19.30–20.30 interval compared to the 07.30–08.30 interval (p<0.005).3 Animals of the adult methylcellulose treated group were lost due to technical problems. Analysis of the time spent in the awake state across the four consecutive time points  revealed = 4.587, p<0.05), without treatment  = 0.115, NS) and age x treatment effect  = 0.034, NS)  = 6.794, p<0.0001) and time x age x treatment interactions  = 3.481, p<0.05) were significant. Two-way ANOVA for each interval separately showed that the time x age x treatment interaction was due to the 17.30–18.30 interval: a significant age x treatment  = 5.455, p<0.05) effect and age effect  = 17.226, p<0.05) was obtained. A Student t-test for the 17.30–18.30 interval showed that fluoxetine decreased non-REM sleep in adult  = 2.732, p<0.05), but not adolescent  = 0.657, NS), rats. The age x treatment interaction for the 19.30–20.30 interval just missed significance  = 4.151, p = 0.054). Finally, there was a significant time effect  = 22.005, p<0.0001), and post-hoc testing indicated that time in non-REM sleep was significantly lower in the 17.30–18.30 interval (p<0.005) and significantly higher in the 19.30–20.30 interval (p<0.005) compared to the 05.30–06.30 and 07.30–08.30 intervals. There was no difference between the later two intervals.Non-REM sleep analysis revealed an overall age effect  . Further = 2.717, NS) and treatment  = 0.553, NS) effects, but the overall age x treatment interaction was significant  = 4.689, p<0.05)  = 3.780, p<0.05). Subsequent analysis revealed a strong trend for an age x treatment interaction during the 19.30–20.30 interval  = 4.264, p = 0.051). No time effect for spindles was observed  = 0.537, NS).Regarding the time spent in spindles, there were no overall age (F p<0.05) . Further = 0.320, NS), treatment  = 0.604, NS) and age x treatment  = 0.598, NS) effects  = 3.615, p<0.05), and a subsequent two-way ANOVA test for the separate intervals showed age effects for the 17.30–18.30  = 13.289, p<0.05) and 07.30–08.30  = 5.232, p<0.05) intervals: REM sleep is reduced in adolescent rats in the 17.30–18.30 interval, and increased in the 07.30–18.30 interval. Finally, a significant effect for time (independent of age or treatment) was obtained  = 11.406, p<0.05), and post-hoc testing revealed that time in REM sleep was significantly lower in the 17.30–18.30 and 07.30–08.30 intervals (p<0.005) compared to the 19.30–20.30 and 05.30–06.30 intervals.Overall REM sleep analysis did not reveal age  = 0.035, NS), and fluoxetine treatment  = 0.021, NS;  = 0.085, NS). Likewise, in the mPFC there were no treatment  = 0.576, NS) and age  = 0.043, NS) effects, and there was no significant age x treatment interaction  = 1.276, NS; 1A receptor immunostaining was decreased in adults rats compared to adolescent rats  = 39.566, p<0.0001), but there were no treatment  = 3.239, NS) or age x treatment interaction  = 1.236, NS; The relative optical density (O.D.) of 5-HT = 7.524, p<0.05), but no treatment effect  = 2.972, NS) or age x treatment interaction  = 0.003, NS) was observed  = 2.977, NS), treatment  = 0.245, NS), and age x treatment  = 0.66, NS) effects were found  = 6.123, p<0.05;  = 0.328, NS) and treatment  = 0.227, NS) effects were found. A subsequent Student t-test revealed that fluoxetine tended to increase PSA-NCAM immunoreactivity in adolescent rats  = 1.503, NS) and tended to decreased it in adult rats  = 1.802, NS), but no significant effects were obtained.PSA-NCAM immunoreactivity in the dorsal raphe nucleus was lower in adult compared to adolescent rats  explain the differential outcomes. Finally, Norcross and colleagues Our findings correspond to the findings of Oh and colleagues While it has been well established that SSRIs decrease REM sleep in both humans and rats 1A, 5-HT1B, and 5-HT2A receptors 1A receptor immunoreactivity in the amygdala. Although PPI is mediated by several other brain areas, it could be one reason why fluoxetine failed to significantly affect PPI.There was a significant decrease in basal startle response in the fluoxetine-treated adult rats compared to the adult controls, an effect that was not found in the adolescent rats. Shanahan and colleagues versus adult 1.04 ng/ml/gram body weight fluoxetine). In line with this, it has been shown that pediatric brain levels of fluoxetine are not significantly different from typical adult levels when corrected for the effects of dose per mass 5 Hrs after the last fluoxetine injection, the fluoxetine concentration in blood plasma was 260±14 ng/ml in adolescents and 375±38 in adults, which falls in the range of a low 50–60 to a high 400–500 ng/ml reported in humans 1A receptor immunostaining in the dorsal raphe nucleus, amygdala and mPFC was not differentially affected by fluoxetine treatment. This finding was somewhat surprising, given the role of the 5-HT1A receptor in the antidepressant effects of SSRIs 1A receptor activation and PSA-NCAM immunoreactivity 1A receptor, because we did not assess 5-HT1A receptor function. We did observe that 5-HT1A immunostaining was lower in adult rats compared to adolescents in the amygdala, which may be in line with the region-independent decline in 5-HT1A receptor immunoreactivity in mice and human during ageing 3 receptor colocalizes with PSA-NCAM in the prefrontal cortex, and that the 5-HT3 receptor antagonist ondansetron reversed the effects of chronic fluoxetine treatment on PSA-NCAM expression in the mPFC 3 receptor is an important target for future research.As reported previously A possible limitation of the present study is that we used commercial animals, both the adolescent and adult rats were shipped 4 days before the start of the fluoxetine treatment. It is conceivable that the adolescent rats were more shipping stress sensitive than adult rats. Yet, given that the adolescent were more active in the Phenotyper, were less immobile in the forced swim test and showed a reduced acoustic startle response compared to adult rats it is not a likely confounding factor. Nonetheless, having more time for acclimatisation following shipping would have been more ideal. Further, because we conducted two behavioral tests in the same group of animals see  it is poVice versa, the anorexic effects of fluoxetine were not seen in adolescent rats. These age-dependent effects of fluoxetine on emotional behavior are most likely due to neuroplastic changes, since amygdalar PSA-NCAM was decreased in adults, but increased in adolescent rats. Together, the data importantly contribute to the debate about the safety of SSRIs in adolescents and the experiment set-up as used here may help in the identification of the mechanisms underlying the age-dependent effects of fluoxetine.In conclusion, we show that adolescent fluoxetine exposure can lead to an adverse increased depression-like outcome. In addition, adolescents may not benefit from fluoxetine's effects on wakefulness.


Beta-hydoxy-beta-methyl butyrate (HMB) when given over a two-week period of time (loading phase) has been demonstrated to decrease skeletal muscle damage, and improve recovery. However, few studies have investigated its acute effects on muscle damage and recovery. Therefore the purpose of this investigation was to determine the effects of short term free acid HMB (HMB-FA) supplementation on serum indices of muscle damage and perceived recovery following a high volume, muscle damaging training session.Twenty resistance trained males aged 21.3 ± 1.9 years with an average squat, bench press, and deadlift of 1.7± 0.2, 1.38 ± 1.9 and 2.07 ± 2.7 times their bodyweight were recruited for the study. Two weeks prior to and throughout the study subjects were placed on a diet consisting of 25 % protein, 50 % carbohydrates, and 25 % fat by a registered dietician who specialized in sport . All subjects participated in a high volume resistance training session consisting of 3 sets of full squats, bench press, deadlifts, pull-ups, bent over rows, shoulder press, barbell curls and triceps extensions. Prior to the exercise session subjects were randomly assigned to receive either a 3 g per day of HMB-FA (Combined with Food-grade orange flavors and sweeteners) or a placebo (Food-grade orange flavors and sweeteners) divided equally into servings given 30 minutes prior to exercise and with two separate meals on day 1. They were then instructed to consume the same amount of HMB-FA or placebo divided into breakfast, lunch and dinner on day two. Immediately prior to the exercise session and 48 hours post exercise, serum creatine kinase (CK), testosterone, cortisol, and perceived recovery scale (PRS) measurements were taken. Perceived Recovery Status consists of values between 0-10, with 0-2 being very poorly recovered with anticipated declines in performance, 4-6 being low to moderately recovered with expected similar performance, and 8-10 representing high perceived recovery with expected increases in performance. A 2 X 2 repeated measures ANOVA was used to assess any main effects. If any main effects were found LSD post hoc tests were incorporated to determine where those differences were located.Significant time and group X time effects were found for CK, which increased to a greater extent in the placebo (140.7 ± 40.9 to 603.8 ± 249.0) than HMB-FA group (158.0 ± 50.9 to 322.2 ± 115.9) (p<0.05). There were also significant time and group X time effects for PRS, which decreased to a greater extent in the placebo (9.1 ± 1.2 to 4.6 ± 1.4) than the HMB-FA group (9.1 ± 0.9 to 6.3 ± 0.9) (p<0.05). There were no time or group X time effects for testosterone or cortisol.These results suggest that an HMB-FA supplement given over a short period of time (48 hours), and without a loading phase to resistance trained athletes can blunt increases in muscle damage and prevent declines in perceived readiness to train following a high volume, muscle damaging resistance training session.