In vitro studies on the binding, antioxidant, and cytotoxic actions of punicalagin

The protective bioactivity of punicalagin, a high molecular weight polyphenol isolated from pomegranate fruit pith and carpellary membrane, against oxidative damages to lipids, amino acids constituting the proteins, and guanosine as a model for DNA has been investigated. The ABTS*-, guanosine, and tryptophan radical generated pulse radiolytically were repaired by punicalagin, k = (0.9-15) x 10(7) dm3 mol-1 s-1. The results are rationalized on the basis of the scavenging activity of punicalagin against various one-electron oxidizing radicals, namely, .OH, N3., and NO2. . The formation of the transient species in these reactions and the rate constants of the scavenging reactions have been probed using a time-resolved kinetic spectrophotometric technique. The antioxidant action of punicalagin is expressed not only through its scavenging reactions but also by its ability to form metal chelates. Binding of punicalagin with bovine serum albumin and metal ions such as iron and copper revealed different binding affinities, whereas its binding with DNA was very weak and nonspecific. In vitro cytotoxic studies against three cell lines, namely, Vero (normal African green monkey kidney cell line), Hep-2 (human larynx epithelial cancer cell line), and A-549 (human small cell lung carcinoma cell line) showed that this polyphenol is toxic only at higher concentration.     

Non-ideal Behavior During Complete Dissolution of Organic Immiscible Liquid: 2. Ideal Porous Media

Column experiments were conducted using ideal natural sands and stainless-steel beads to examine the complete dissolution behavior of an organic immiscible liquid. Trichloroethene was used as the representative organic liquid. The elution curves exhibited multi-step behavior, with multiple extended periods of relatively constant contaminant flux. These secondary steady-state stages occurred at concentrations several orders-of-magnitude below aqueous solubility for the well-sorted sands. In contrast, the secondary steady-state stages occurred within 1 log of aqueous solubility for the poorly sorted sand. The non-ideal behavior is hypothesized to result from constraints to hydraulic accessibility of the organic liquid to flowing water, which may be expected to be mediated by the pore-scale configuration of the flow field and the fluid phases.     

A small-molecule modulator of poly-alpha 2,8-sialic acid expression on cultured neurons and tumor cells

Poly-alpha2,8-sialic acid (PSA) has been implicated in numerous normal and pathological processes, including development, neuronal plasticity, and tumor metastasis. We report that cell surface PSA expression can be reversibly inhibited by a small molecule, N-butanoylmannosamine (ManBut). Inhibition occurs through a metabolic mechanism in which ManBut is converted to unnatural sialic acid derivatives that effectively act as chain terminators during cellular PSA biosynthesis. N-Propanoylmannosamine (ManProp), which differs from ManBut by a single methylene group, did not inhibit PSA biosynthesis. Modulation of PSA expression by chemical means has a role complementary to genetic and biochemical approaches in the study of complex PSA-mediated events.     

Influence of Apoptosis in Bovine Embryo's Development

The correlation between apoptosis and early bovine embryonic loss is still not fully elucidated. In the present study, the relationship between the arrest of bovine embryos at the different stages of development and apoptosis was evaluated. We used embryos 7 days after in vitro maturation and fertilization, and morphologic and biochemical apoptotic analyses were performed by using a phase contrast microscope and by the terminal transferase dUTP nick end‐labelling respectively. For the statistic, the apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. To evaluate the relation between ACR and fragmentation pattern, embryos were divided into five groups, groups I–V. To assess the relation between ACR and cytoplasmatic fragmentation, embryos were divided into three groups, according to the fragmentation percentage (<5%; 5–15% and >15%). Of the total 139 embryos included, 65 arrested at 2–8 cells; 14 arrested at 9–16 cells; 18 compacted morula and 42 were non‐arrested blastocysts. The average number of embryonic fragmentation at different stages of the development, 2–8 cells, 9–16 cells, compacted morula and blastocyst, was 16.0 ± 1.5, 28.7 ± 4.4, 4.4 ± 2.4 and 1 ± 0.3 respectively. The embryos at the stage of arrested 9–16 cells and compacted morula had higher ACR than those at the blastocyst stage, excluding the stage of 2–8 cells (the genome is not yet active). The correlation detected between embryonic development and ACR was 0.92 (p < 0.01). It was observed that embryos possessing high fragmentation showed the higher ACR value (r = 0.98, p < 0.05). Comparing the results between fragmentation percentage and ACR, it was observed that the embryos with higher percentage of fragmentation corresponded to higher ACR (r = 0.97, p < 0.01). These results clearly demonstrated that bovine embryonic arrest at different stages of development is correlated with the apoptotic mechanisms.     

Ameliorative Effects of Melatonin against Hydrogen Peroxide‐Induced Oxidative Stress on Boar Sperm Characteristics and Subsequent In Vitro Embryo Development

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H₂O₂) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ). The sperm were treated with melatonin in the presence or absence of H₂O₂ for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H₂O₂ groups were lower than H₂O₂ only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.     

Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co‐culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide‐induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl‐2, Caspase‐3 and Bax) by RT‐PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co‐cultured with BOEC pre‐treated with astaxanthin (500 μm ) in the presence or absence of sodium nitroprusside (SNP, 1000 μm ) for 24 h. Cell viability in BOEC treated with SNP (50–2000 μm ) lowered, while astaxanthin addition (50–500 μm ) increased it in a dose‐dependent manner. Cell viability in astaxanthin plus SNP (1000 μm ) gradually recovered according to the increase in astaxanthin additions (100–500 mm ). The LPO in astaxanthin group (50–500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT‐PCR. Bcl‐2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase‐3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6–7 days under co‐culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μm astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.     

Persistent Mullerian Duct Syndrome in a Miniature Schnauzer Dog with Signs of Feminization and a Sertoli Cell Tumour

A 5‐year‐old male Miniature Schnauzer was presented with unilateral cryptorchidism and signs of feminization. Abdominal ultrasonography revealed an enlarged right testis and a large, fluid‐filled cavity that appeared to arise from the prostate. Computed tomography revealed the cavity to be consistent with an enlarged uterine body, arising from the prostate, and showed two structures resembling uterine horns that terminated close to the adjacent testes. The dog had a normal male karyotype, 78 XY. Gonadohysterectomy was performed and both the surgical and the histological findings confirmed the presence of a uterus in this male animal, resulting in a diagnosis of persistent Mullerian duct syndrome (PMDS). The enlarged intra‐abdominal testis contained a Sertoli cell tumour. Computed tomography proved to be an excellent diagnostic tool for PMDS.     

RESEARCH PAPER: Segmental dorsolumbar epidural analgesia via the caudal approach using multiple port catheters with ketamine or lidocaine or in combination in cattle

ObjectiveTo determine the analgesic and systemic effects of epidural administration of ketamine, lidocaine or a combination of ketamine/lidocaine in standing cattle.Study designProspective, randomized, experimental trial.AnimalsSix healthy male cattle weighing between 335 and 373 kg.MethodsThe animals received 0.5 mg kg^?1 of ketamine (K), 0.2 mg kg^?1 of 2% lidocaine (L) or 0.25 mg kg^?1 ketamine plus 0.1 mg kg^?1 lidocaine (KL). All the drugs were injected into the dorsolumbar epidural space via a caudal approach through a non‐styletted multiple‐port catheter. Each animal received each treatment at random. Evaluations of analgesia, sedation, ataxia, heart rate, arterial pressure, respiratory rate, skin temperature and rectal temperature were obtained at 0 (basal), 5, 10, 15, 30, 45, 60, 75, 90 minutes after epidural injection, and then at 30‐minute intervals until loss of analgesia occurred. Skin temperature was taken at these intervals up to 60 minutes. All the animals received a standard noxious stimulus; a 4‐point scale was used to score the response. A second scale was used to score ataxia and a third for sedation.ResultsThe duration of analgesia in the upper and lower flanks in cattle was 140 ± 15, 50 ± 14 and 80 ± 22 minutes (mean ± SD) after dorsolumbar epidural KL, K or L, respectively. The cardiovascular changes were within acceptable limits in these clinically healthy cattle.ConclusionsDorsolumbar epidural administration of KL to cattle resulted in longer duration of analgesia of the upper and lower flanks in standing conscious cattle, than the administration of K or L alone.Clinical relevanceFurther research is necessary to determine whether this combination using this technique provides sufficient analgesia for flank surgery in standing cattle.     

Isolation and Culture of Ovine and Bubaline Small and Large Pre-antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle

Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 ± 3.3 vs 12.1 ± 2.6 and LPFs: 16.0 ± 3.6 vs 9.2 ± 1.8) and buffaloes (SPFs: 9.2 ± 1.3 vs 4.1 ± 1.0 and LPFs: 10.3 ± 2.7 vs 5.4 ± 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 ± 2.7 vs 16.4 ± 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.     

Enhanced and delayed stress-induced alcohol drinking in mice lacking functional CRH1 receptors

There is a relation between stress and alcohol drinking. We show that the corticotropin-releasing hormone (CRH) system that mediates endocrine and behavioral responses to stress plays a role in the control of long-term alcohol drinking. In mice lacking a functional CRH1 receptor, stress leads to enhanced and progressively increasing alcohol intake. The effect of repeated stress on alcohol drinking behavior appeared with a delay and persisted throughout life. It was associated with an up-regulation of the N-methyl-d-aspartate receptor subunit NR2B. Alterations in the CRH1 receptor gene and adaptional changes in NR2B subunits may constitute a genetic risk factor for stress-induced alcohol drinking and alcoholism.