Suppression of plasminogen activator inhibitor-1 (PAI-1) activity by crocin ameliorates lipopolysaccharide-induced thrombosis in rats.

The imbalance between clot formation and fibrinolysis is mainly attributed to increased levels of plasminogen activator inhibitor type 1 (PAI-1), an inhibitor of fibrinolysis closely involved in inflammatory responses such as septic shock. This increase is mediated by many factors, including reactive oxygen species (ROS). The present study was designed to evaluate the prophylactic effect of crocin, a potent natural antioxidant, on PAI-1 in the rat model of endotoxic shock. Lipopolysaccharide-infused rats (500 µg/kg) showed significant changes in thrombosis-related haematological parameters such as decrease of platelet blood counts and increase (7 fold) of PAI-1 concentration in blood plasma. No effect on t-PA activity was observed. Crocin administration in two different doses (10 mg/kg and 100 mg/kg) 30 min prior to the injection of LPS, inhibited the reduction of platelet counts and ameliorated the concentration of PAI-1 in the liver and the brain. Moreover, crocin inhibited the deposition of fibrin in the renal glomeruli. No significant changes were recorded in the healthy groups of crocin (10 mg/kg and 100 mg/kg) compared to the control group. These data demonstrate the potential of crocin to prevent LPS-induced organ injury and suggest it is worthwhile to investigate the use of antioxidants for the treatment of septicemia.

Dietary omega-3 polyunsaturated fatty acids induce plasminogen activator activity and DNA damage in rabbit spermatozoa.

The aim of this study was to determine the effect(s) of dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) on rabbit semen. Adult rabbit bucks were assigned to two groups that were given two diets, a standard diet (control) and a diet supplemented with ω-3 PUFA. Sperm samples were collected from all bucks with the use of an artificial vagina in 20-day intervals, for a total period of 120 days. The enrichment of membranes in ω-3 PUFA was manifested by the elevation of the 22:5 ω-3 (docosapentaenoic acid [DPA]) levels within 40 days. This increase in DPA content did not affect semen characteristics (i.e., concentration, motility and viability). However, it was associated with the induction of lipid peroxidation in spermatozoa, as determined on the basis of the malondialdehyde content. Lipid peroxidation was associated with DNA fragmentation in ω-3 PUFA-enriched spermatozoa and a concomitant increase in plasminogen activator (PA) activity. The effects of ω-3 PUFA on sperm cells were evident within 40 days of ω-3 PUFA dietary intake and exhibited peack values on day 120. Our findings suggest that an ω-3 PUFA-rich diet may not affect semen characteristics; however, it may have a negative impact on the oxidative status and DNA integrity of the spermatozoa, which was associated with an induction of PAs activity.

Protective effect of crocetin on bovine spermatozoa against oxidative stress during in vitro fertilization.

Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of in vitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5 µm), for 120 or 240 min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5 µm). The results indicate that incubation of spermatozoa with 2.5 µm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p < 0.05). The presence of crocetin (2.5 µm) in the fertilization medium also resulted in a significant increase in acrosome-reacted spermatozoa and blastocyst production, compared to the control group (p < 0.01). These data indicate that crocetin (2.5 µm) positively affects bovine sperm quality characteristics during a 240-min incubation and improves its fertilizing ability, directly and/or indirectly, by regulating ROS concentration and lipid peroxidation.

Seasonal variation of plasminogen activator activity in spermatozoa and seminal plasma of boar, buck, bull and stallion.

Plasminogen activators (PA) are proteolytic enzymes present in the spermatozoa and seminal plasma of various species. They play a role in the binding of the spermatozoon and its penetration through the layers surrounding the oocyte. Plasminogen activator activity (PAA) is modulated by hormones that have a seasonal variation, such as testosterone and melatonin. The present study investigates the seasonal variation of PA activity in sperm extracts and seminal plasma of four farm animal species: boar, buck, bull and stallion. Semen samples were collected every second week during a 12-month period and PAA was determined. With respect to sperm enzyme activity, the boar showed a peak from late January until the beginning of April, whereas the activity in the bull was at the highest levels from April until October and gradually declined during autumn and winter period. Plasminogen activator activity of stallion spermatozoa peaked during March and April, and remained low throughout the rest of the year, whereas in the buck sperm, PAA increased from late October until the end of January. No biologically significant variation was detected regarding the seminal PAA activity in any of the species studied. While seasonality of reproduction is typically studied from the female perspective, the present data provide compelling information about a factor that may affect the reproductive ability of the male.

Effect of long-term dietary administration of oregano on the alleviation of carbon tetrachloride-induced oxidative stress in rats.

This study aimed at evaluating the protective effect of long-term dietary oregano on the alleviation of carbon tetrachloride-induced oxidative stress in rats. Twenty-four female Wistar rats were allocated to four groups of six animals each. Groups 1 (control) and 2 (CCl 4) were fed a basal diet, while groups 3 (oregano) and 4 (oregano + CCl 4) were fed the basal diet supplemented further with ground oregano at 1% level. Following six-week feeding, the rats of groups 2 and 4 were given a single intraperitoneal injection of CCl 4 at a dose of 1 mL/kg bw. Six hours after the CCl 4 injection, all animals were sacrificed, and serum, liver, kidney, and heart tissue samples were collected. Analysis results showed that the addition of oregano significantly increased the total phenolic content and the Trolox equivalent antioxidant capacity of the basal diet but had no effect on its lipid peroxidation index. Treatment with CCl 4 of rats from the CCl 4 group caused a significant increase in aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) in serum, whereas it decreased cholesterol and triglyceride content as compared to the control. It also increased the lipid peroxidation index and decreased the scavenging activities of the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS) radical cation, the hydroxyl anion radical, the superoxide anion radical, and the hydrogen peroxide in all tested tissues, as compared to that of the control. Without CCl 4 treatment, diet supplementation with oregano had no effect on these biochemical parameters, excluding the hydroxyl radical scavenging activity, which was increased in all tested tissues as compared to that of the control. Feeding oregano before CCl 4 treatment resulted in a significant decline of the increase in AST, ALT, and ALP activities ( P < 0.05 vs CCl 4 group), but the recorded values could not attain those of the control group ( P < 0.05 vs control group). It significantly increased the reduced cholesterol and triglycerides ( P < 0.05 vs CCl 4 group) to values not differing from those of the control. It also resulted in a significant reduction of the increased malondialdehyde ( P < 0.05 vs CCl 4 group) to values that could not attain the levels of the control but had no significant effect ( P > 0.05) on the reduced ABTS radical cation scavenging activity. It increased significantly the reduced hydroxyl anion radical scavenging activity ( P < 0.05 vs CCl 4 group) to values that could not attain those of the control in all tested tissues except kidney. Additionally, it resulted in a significant elevation of the decreased superoxide anion radical scavenging activity in serum and liver but had no effect in kidney and heart, whereas it also resulted in a significant elevation of the decreased hydrogen peroxide scavenging activity in liver, kidney, and heart but had no effect in serum. These results suggest that dietary oregano may effectively improve the impaired antioxidant status in CCl 4-induced toxicity in rats.

Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione.

Reactive oxygen species (ROS) are mediators of lung injury, and glutathione (GSH) is the major nonprotein antioxidant that protects the cell from oxidative stress. We have recently shown that H(2)O(2) induces ceramide-mediated apoptosis in human lung epithelial cells. We hypothesized that ROS-mediated depletion of GSH plays a regulatory role in ceramide generation, and thus in the induction of apoptosis. Our present studies demonstrate that GSH at physiologic concentrations (1 to 10 mM) inhibits ceramide production in a time- and dose-dependent manner in A549 human alveolar epithelial cells. On the other hand, buthionine-sulfoximine-mediated depletion of intracellular GSH induces elevation of ceramide levels and apoptosis. In addition, GSH blocks H(2)O(2)-mediated induction of intracellular ceramide generation and apoptosis. These effects were not mimicked by oxidized GSH (GSSG) or other thiol antioxidants, such as dithiothreitol and 2-mercaptoethanol. Moreover, increase of intracellular H(2)O(2), mediated by inhibition of catalase by aminotriazole, also induces ceramide generation and apoptosis. These effects were blocked by N-acetylcysteine. Our results suggest that GSH depletion may be the link between oxidative stress and ceramide-mediated apoptosis in the lung.

Contribution of cysteine 158, the glycosylation site threonine 194, the amino- and carboxy-terminal domains of apolipoprotein E in the binding to amyloid peptide beta (1-40).

Recent studies have shown that at physiological conditions (pH 7.6, 37 degrees C), the reactivity of recombinant apoE isoforms secreted by mammalian cells toward amyloid peptide beta (Abeta40) follows the order apoE2 > apoE3 > apoE4 for the apoE monomer and apoE2 > apoE3 for apoE dimer that is formed via that intramolecular disulfide bridges. Different Abeta binding properties have been reported for the plasma-derived apoE and commercially available apoE preparations that differ from the native apoE forms in the degree of their O-glycosylation. To define structural elements of apoE involved in the interaction with Abeta, we have introduced point mutations as well as amino- and carboxy-terminal deletions in the apoE structure. The mutant apoE forms were expressed transiently using the Semliki Forest Virus system, and the culture medium was utilized to study the reactivity of the mutated proteins with Abeta 40. This analysis showed that a mutation in the O-glycosylation site of apoE2 (Thr194-Ala) did not affect the SDS-stable binding of apoE to Abeta. In contrast, introduction of cysteine at position 158 of apoE4 (Arg112, Cys158) increased the SDS-stable binding of apoE to Abeta to the levels similar to those observed in apoE2. Similar analysis showed that apoE truncated at residues 259, 249, 239, and 229 retains the SDS-stable binding to Abeta40, whereas apoE truncated at residues 185 and 165 does not bind to Abeta. The deletion of aminoterminal residues 2-19 reduced the SDS-stable binding of apoE2 to Abeta and deletion of residues 2-81 abolished binding to Abeta. It is also noteworthy that the (Delta2-81) apoE mutant exists predominantly as a dimer, suggesting that removal of residues 2-81 promoted dimerization of apoE. These findings suggest that the amino- and carboxy-terminal residues of apoE are required for SDS-stable binding of apoE to Abeta and that the presence of at least one cysteine contributes to the efficient Abeta binding.

Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors.

Human apolipoprotein CIII (apoCIII) is a major determinant of plasma triglyceride metabolism. The regulatory elements that control both hepatic and intestinal transcription of the human apoCIII gene are localized between nucleotides -792 and -25 of the apoCIII promoter. Elements important for apoCIII promoter activity are three hormone response elements (HREs) and three SP1-binding sites. Orphan members of the nuclear hormone receptor superfamily can bind the HREs and strongly enhance or repress apoCIII promoter activity. In the present study we have investigated the ability of ligand-dependent nuclear hormone receptors to bind and modulate the human apoCIII promoter activity. Experiments using DNA binding and competition assays showed that the proximal element B (-87/-72) binds strongly, in addition to HNF-4, ARP-1, EAR-2, and EAR-3, heterodimers of RXRalpha with RARalpha, and less efficiently, homodimers of RARalpha and heterodimers of RXRalpha with T3Rbeta or PPARalpha. Element G (-669/-648), which was shown previously to bind ARP-1 and EAR-3 but not HNF-4, binds strongly heterodimers of RXRalpha with either RARalpha or T3Rbeta. Finally element I4 (-732/-712), which was shown to bind HNF-4, also binds strongly ARP-1 and EAR-3, as well as RXRalpha/RARalpha heterodimers and less efficiently, RXRalpha/T3Rbeta heterodimers. Methylation interference experiments have identified the protein-DNA interactions between different nuclear receptors and the respective HREs on the apoCIII promoter. RXRalpha/RARalpha heterodimers and HNF-4 homodimers bind to DR-1 motifs on elements B and I4, respectively. RXRalpha/T3Rbeta heterodimers and ARP-1 bind to DR-5 and DR-0 motifs respectively on element G. Cotransfection experiments in HepG2 cells showed that RXRalpha or a combination of RXRalpha and RARalpha increased the apoCIII promoter activity approximately 2-fold in the presence of the ligands 9-cis or all-trans RA. In contrast, a combination of RXRalpha and T3Rbeta transactivated the apoCIII promoter 1.5-fold in the presence of 9-cis RA but it repressed the apoCIII promoter activity in the presence of T3. Mutations in the HREs of elements B, G, or I4 or in the SP1-binding site of element H, which abolished the binding of nuclear hormone receptors or SP1 to their cognate site, reduced the promoter strength and exhibited different responses to the ligand-dependent nuclear receptors. The findings suggest that modulation of the apoCIII promoter activity by orphan and ligand-dependent nuclear receptors involves complex interactions among nuclear receptors, SP1 and possibly other factors bound to the enhancer and the proximal promoter region.

Transactivation of the ApoCIII promoter by ATF-2 and repression by members of the Jun family.

It was shown previously that cytokines such as tumor necrosis factor-alpha that stimulate signal transduction pathways involving transcription factors ATF-2 and Jun repress apoCIII promoter activity in HepG2 cells. In the present study, DNase I footprinting analysis established that ATF-2 protected three regions in the apoCIII promoter. One region (-747/-726) present in the apoCIII enhancer is within the previously identified footprint I and has overlapping boundaries with the binding sites of Sp1 (-764/-742) and HNF-4 (-736/-714). The other two regions represent new footprints and have been designated D/E (-219/-199) and B/C (-102/-75). The B/C region overlaps with the previously identified footprint B which contains an HNF-4 binding site (-87/-63). Cotransfection experiments in HepG2 cells showed that ATF-2 transactivated the -890/+24 apoCIII promoter 1.6-fold. In addition, mutations in the proximal D/E (-219/-199) and distal I (-747/-726) ATF-2-binding sites reduced the apoCIII promoter strength to 33 and 9% of control, respectively, indicating that ATF-2 is a positive regulator of apoCIII gene transcription. Cotransfections with ATF-2 and HNF-4 expression plasmids resulted in additive transactivation of the apoCIII promoter. Furthermore, apoCIII promoter constructs bearing mutations in the D/E and I ATF-2 binding sites were efficiently transactivated by HNF-4, suggesting that these two factors contribute independently to the apoCIII promoter strength. Members of the Jun family (c-Jun, JunB, and JunD) caused a dose-dependent inhibition of the -890/+24 apoCIII promoter activity. A synthetic promoter containing the apoCIII enhancer in front of the minimal AdML promoter was also repressed by Jun. In contrast, apoCIII promoter segments lacking the enhancer region were transactivated by Jun. The findings suggest that homodimers of Jun or heterodimers of Jun with other AP-1 subunits could be responsible for the observed repression by interfering with the function(s) of the apoCIII enhancer. Repression by Jun could be reversed in the presence of ATF-2 and HNF-4, suggesting that ATF2 and possibly Jun/ATF-2 heterodimers exert a positive effect on apoCIII gene transcription, as opposed to Jun homodimers or heterodimers with other AP-1 members. These findings suggest a role for members of the Jun family and ATF-2 that participate in signal transduction pathways in basal or induced apoCIII promoter activity in cells of hepatic origin.

The polytene chromosomes of Drosophila triauraria and D. quadraria, sibling species of D. auraria.

The polytene chromosomes of Drosophila triauraria and D. quadraria, two of the sibling species of D. auraria, were examined. The polytene chromosomes of all three species exhibit very clear homology. Unlike the stock of D. auraria that we studied, D. triauraria and D. quadraria carry heterozygous paracentric inversions. In both species, 2R and 3R are the arms where these inversions are concentrated. In addition, in D. quadraria, the 3L chromosome arm is very complicated because of heterozygous inversions. The mode of inheritance of these rearrangements was studied. A homozygous strain for all chromosome arms of D. triauraria was isolated, while a homozygous strain was obtained only for the arms X, 2L, 3L, and 4 of D. quadraria. Like D. auraria, both species show a large number of inverted tandem duplications in the paired condition, even in the chromosomes of their hybrids. Small deletions were also detected, one of which, in D. triauraria, is homozygous terminal. Hypotheses are discussed concerning the relationships of the species and the existence of inverted tandem duplications.