In vitro expression and inhibition of procoagulant activity produced by bovine alveolar macrophages and peripheral blood cells

Local and systemic activation of coagulation is frequently associated with bacterial sepsis. The coagulopathy is due, at least in part, to expression of tissue factor (TF) by monocytes and macrophages. The purpose of this study was to evaluate the expression of procoagulant activity by bovine alveolar macrophages, leukocytes and platelets, and to determine the relative potency of three chemical inhibitors of TF expression (pentoxifylline, retinoic acid, and cyclosporin A). Bovine alveolar macrophages were stimulated with lipopolysaccharide (LPS) derived from Pasteurella haemolytica or recombinant bovine tumour nervous factor (TNF) and dose- and time-dependent effects on TF expression were studied. LPS and TNF induced TF expression in alveolar macrophages and LPS treatment of whole blood induced TF expression in mononuclear cells. Neutrophils and platelets also expressed procoagulant activity, but this activity was not inhibited by anti-bovine TF monoclonal antibody. Pentoxifylline (40 mol/L), retinoic acid (0.01 mmol/L) and cyclosporin A (0.08 mol/L) inhibited TF expression when added concurrently with LPS or TNF, but not when added 4 h after stimulation. TF mRNA was not detected in unstimulated alveolar macrophages by Northern blot analysis. In contrast, exposure to LPS or TNF for 6 h induced marked expression of TF mRNA, which was inhibited by treatment with pentoxifylline, retinoic acid and cyclosporin A. Expression of TNF by alveolar macrophages stimulated with LPS was also inhibited by these compounds. Our results indicate that procoagulant activity expressed by alveolar macrophages and monocytes is associated with expression of TF, whereas procoagulant activity expressed by neutrophils and platelets is not. The concentrations of pentoxifylline and retinoic acid necessary for inhibition of TF expression in vitro may not be achievable in vivo owing to their toxic effects. However, the in vitro concentration of cyclosporin A that inhibited TF expression did not exceed the plasma concentration observed in humans, and therefore may be useful for inhibition of TF expression in vivo.Abbreviations BAL bronchoalveolar lavage - LPS lipopolysaccharide - cDNA cloned deoxyribonucleic acid - cAMP cyclic adenosine monophosphate - GAPDH glyceraldehyde phosphate dehydrogenase - mRNA messenger ribonucleic acid - TF tissue factor - TNF tumour necrosis factor - DPBS Dulbecco's phosphate-buffered saline     

Independent and combined effects of Satureja khuzistanica essential oils and acetic acid on prevalence and intensity of fatty liver syndrome in broiler chickens

Two hundred fifty two male Ross 308 broiler chicks were raised to examine the effects of Satureja khuzistanica essential oils (SkEO; 0, 200, 300, 400, 500 and 600 mg/bird/day) administrated via oral gavage and dietary supplementation of acetic acid (AA; 0 or 20 g/kg) in a 6 × 2 factorial fashion using a complete randomized block design with seven replicates of three birds each. In Day 34 of age, liver fat percentage decreased by increasing SkEO levels in a quadratic trend (p < .05). Oral gavag of 200 and 300 mg of SkEO decreased AST activity by 16.67% and 16.94%, respectively, compared with the control birds (p < .05). In Day 38 of age, liver percentage was greater by 0.37% in the birds received 400 mg SkEO than those given 500 mg (p < .05). Liver fat percentage decreased in the birds fed the acidified diet and received 300 and 500 mg SkEO as well as in the birds offered 200 and 400 mg of SkEO and fed non-acidified diet than those received acidified diet with no SkEO administration (p < .05). Prevalence of score zero for hepatocytic vacuolations and necrosis was 80.00% and 42.42% in the birds receiving 600 mg SkEO respectively. Median for relative frequency of scores for hepatocytic vacuolations and hepatocytic necrosis was lesser in the birds received SkEO by oral gavage compared with that of control birds (p < .05). In conclusion, administration of SkEO via oral gavage reduced liver fat in broiler chicken but no consistent dosage could be verified as the effective dosage for all ages concerned. The acidified diet per se showed no evident effect on liver fat at all ages considered. The SkEO × AA interaction exhibited promising but unreliable effects on liver colour score in market age (42 days).     

Recovery of sperms bearing X chromosomes with different concentrations of magnetic nanoparticles in ram

Pre-conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non-toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 μg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa- bearing X chromosomes with 50 μg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 μg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 μg/ml MNP significantly reduced DNA integrity.     

Molecular characterisation of Theileria equi and risk factors associated with the occurrence of theileriosis in horses of Punjab (Pakistan)

Theileria equi (T. equi) is an obligate intra- and extra-erythrocytic parasite that causes equine theileriosis (ET) in equids. Equine theileriosis is considered a notifiable disease of global significance, a major constraint to the international movement of horses, and endemic in many countries. This disease may be difficult to diagnose, as it can produce variable and nonspecific clinical signs. A cross-sectional study was designed for the molecular characterisation of T. equi and to investigate the associated risk factors of ET accompanied by its consequences on haematological and sero-biochemical parameters. A convenience sampling of 500 blood samples were collected from ET suspect horses from January to December 2017. PCR was performed on all blood samples targeting the 18S rRNA gene of T. equi followed by sequencing; 9% animals tested positive with confirmed sequences. The isolates of this study showed high homology with Cuban, Russian and Brazilian isolates of T. equi (accession numbers KY111762.2 , MG551915.1 and KY952237.1 , respectively). Based on multivariate analysis, the principal risk factors consisted of absence of dogs on the premises and presence of tick infestation. The haemato-biochemical parameters showed a decrease in granulocytes and erythrocytes, and an increase in lymphocytes, monocytes, mean corpuscular volume, mean corpuscular haemoglobin, mean platelet volume, glucose, phosphorus and aspartate aminotransferase in positive horses. This is the first study which identified ET in Punjab (Pakistan) using molecular techniques and risk factors together with the haemato-biochemical variations in horses.     

Temporal variation of total and DTPA-extractable heavy metal contents as influenced by sewage sludge and perlite in a calcareous soil

Continuous application of sewage sludge (SS) as a source of organic matter, may increase available heavy metals (HMs) up to their critical levels. As a consequence their uptake by plants, risks of accumulation in food chain and groundwater pollution increases. To evaluate the effects of perlite and SS on total content and available fraction of iron (Fe), manganese (Mn), lead (Pb), copper (Cu), nickel (Ni), cadmium (Cd) and zinc (Zn) over 45 and 90 days a completely randomized pot experiment was conducted. Treatments consisted of SS (0, 2 and 4%) and combined SS and perlite levels (1:1, 1.5:0.5 and 0.5:1.5% SS:perlite). Mean available Fe, Ni, Cd, Mn and Zn fractions decreased over time by 7–27%, whereas, total contents did not change. Available HMs in SS-treated soils increased by 0.5–25%; whereas, only total Zn, Cu and Pb concentrations increased with SS application (although concentrations fall in the range of critical levels). The lowest (1.6 fold) and highest (22.1 fold) increases obtained for available Cu and Zn, respectively. Perlite could mitigate the adverse effect of high rate application of SS on amplification of HMs extractability. Therefore, their concomitant applications can be suggested considering the perlite durability and its positive effects on soil physical properties.     

Immobilization of Urease onto Modified Egg Shell Membrane through Cross Linking

Background:Immobilization is an approach in industry to improve stability and reusability of urease. The efficiency of this technique depends on the type of membrane and the method of stabilization. Methods:The PEI-modified egg shell membrane was used to immobilize urease by absorption and glutaraldehyde cross-linking methods. The membranes were characterized by FTIR and AFM, and Nessler method was applied to measure the kinetic of the immobilized enzymes. Finally, the storage stability (6 °C for 21 days) and reusability (until enzyme activity reached to zero) of the immobilized enzymes were investigated. Results:Based on FTIR, three new peaks were observed in both the absorption- (at 1389.7, 1230.8, and 1074.2 cm^-1) and the cross-linking (at 1615-1690, 1392.7, 1450 cm^-1) immobilized enzymes. The surface roughness of the native membrane was altered after PEI treatment and enzyme immobilization. The optimal pH of cross-linking immobilized enzymes was shifted to a more neutral pH, while it was alkaline in adsorption-immobilized and free enzymes. The reaction time decreased in all immobilized enzymes (100 min for free enzyme vs. 60 and 30 min after immobilizing by adsorption and cross-linking methods, respectively). The optimal temperature for all enzymes was 70 °C and they had a higher K_m and a lower V_max than free enzyme. The stability and reusability of urease were improved by both methods. Conclusion:Our findings propose these approaches as promising ways to enhance the urease efficiency for its applications in industries and medicines. Key Words: Egg shell, Immobilization, Polyethylenimine, Urease     

Investigating the reduction of sodium adsorption ratio from agricultural waste by rice husk in batch scale and physical model of drain envelop

Sodium adsorption ratio (SAR) is one of the water quality indexes that whose is important due to reuse or depletion to environment. Solutes in drain water can be controlled by adsorption, chemical or biological reaction, organic envelope of drainage. Rice husk is the common option of drainage envelops in paddy fields. In this study, the ability of reduction of SAR by rice husk was evaluated in batch scale and physical model of drain envelops. In the batch experiments, the adsorption of SAR parameters was investigated by adding 2 g of rice husk into a 100 ml of sodium chloride solution. The results indicated that rice husk absorbed calcium, magnesium and sodium, respectively. By increasing the temperature, contact time and pH, adsorption of calcium, magnesium and sodium was increased; however, the higher concentration of sodium in soil solution reduced the percentage of adsorption. In a more realistic state, physical models of subsurface drainage in the paddy fields were made. Drainage envelope treatments included of rice husk (H), combination of 20 and 60 % of husk with gravel (H₂₀G₈₀ and H₆₀G₄₀) and a pipe without envelope (NE). Due to higher drain discharge and more sodium removal (lower SAR in drain water), treatment H with the discharge of 16.2 ml/min and SAR of 1.27 (meq/l)^0.5 was better in comparison with other treatments.     

Chemical Constituents of Lagenaria siceraria Mesocarp and Its Xanthine Oxidase and Alpha-Amylase Inhibitory Activities

The present study was conducted to identify natural products in the bioactive fractions of Lagenaria siceraria mesocarp. Column chromatography and gas chromatography-mass spectrometry (GC-MS) was used for separation and identification of phytochemicals. Methanolic extract and its fractions displayed alpha-amylase and xanthine oxidase inhibitory activities comparable to standard drugs. GC-MS analysis of the fractions and column eluates revealed the presence of a variety of chemical compounds, including 4-(methoxymethyl)phenol, 2,2?-methylenebis{6(1,1-dimethylethyl)-4-ethyl}phenol, methyl 2-hydroxy-3-phenylpropanoate, oxacyclododecane-2,8-dione, N-(2-(4-hydroxyphenyl)ethyl] acetamide, 2,3-Epoxycarane, 4-(propan-2-yl)benzaldehyde, octadec-1-ene, 4a-methyl-1-methylidene-1,2,3,4,4a,9,10,10a-octahydrophenanthrene, 2,6-di(propan-2-yl)naphthalene, methyl 3-(4-hydroxy-3,5-dimethylphenyl)propanoate, 4,8a-dimethyl-6-(prop-1-en-2-yl)-3,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one 1,3-dihydro-2-benzofuran, methyl N-hydroxybenzenecarboximidoate, linoleic acid, and palmitic acid. Many of these compounds are being reported for the first time from this plant, the presence of which may explain, at least partly, its medicinal efficacy. The fruit possesses significant anti-enzymatic properties against alpha-amylase and xanthine oxidase, and the most active compounds of mesocarp of the fruit appear in ethyl acetate fraction of its methanolic extract.     

Effects of carbohydrate source and polyethylene glycol on maturation and germination of somatic embryos in walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Low efficiency of somatic embryo maturation, germination, and conversion to plantlets is a major problem in many species including J. regia L. Germination efficiency of somatic embryos is very low in walnut. In this study, effects of two carbohydrate sources, sucrose and maltose (each at 3% and 6%), and two kinds of PEG (4000 and 6000) (each at four levels of 1.5%, 3%, 5% and 7.5%) on maturation and germination of walnut embryos were tested. The number of somatic embryos increased conspicuously on medium containing PEG. Furthermore, higher levels of PEG-4000 (7.5%) could remarkably enhance the morphogenesis of somatic embryos and the number of embryos produced. PEG-4000 stimulated somatic embryo maturation of walnut. This stimulatory effect was dependent on the carbohydrate source used. Sucrose in combination with PEG-4000 produced 50% of cotyledonary and normal somatic embryos. Different concentrations of PEG were effective on the number of embryos with a shoot meristem. PEG-4000 7.5% and sucrose 3.0% produced the highest rate (50.0%) of normal shooting embryos. However, PEG (4000, 6000) and maltose caused an unfavorable effect and increased the frequency of abnormal shaped somatic embryos.