Minimizing the central hydrophobic domain in oleosin for the constitution of artificial oil bodies

Oleosin, a unique structural protein anchoring onto the surface of seed oil bodies by its central hydrophobic domain, stabilizes these lipid-storage organelles as discrete entities. Stable artificial oil bodies have been successfully constituted with native or recombinant oleosins. In this study, recombinant sesame oleosin with 12 residues stepwise truncated from its central hydrophobic domain of 72 residues was overexpressed in Escherichia coli, was purified to homogeneity, and was used for the constitution. Artificial oil bodies constituted by truncated oleosins with the central hydrophobic domain longer than 36 residues were as stable as native sesame oil bodies, and those constituted by truncated oleosins lacking more than half of the original central hydrophobic domain inclined to coalesce upon collision or aggregation.     

Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

Essential role of Fkbp6 in male fertility and homologous chromosome pairing in meiosis

Meiosis is a critical stage of gametogenesis in which alignment and synapsis of chromosomal pairs occur, allowing for the recombination of maternal and paternal genomes. Here we show that FK506 binding protein (Fkbp6) localizes to meiotic chromosome cores and regions of homologous chromosome synapsis. Targeted inactivation of Fkbp6 in mice results in aspermic males and the absence of normal pachytene spermatocytes. Moreover, we identified the deletion of Fkbp6 exon 8 as the causative mutation in spontaneously male sterile as/as mutant rats. Loss of Fkbp6 results in abnormal pairing and misalignments between homologous chromosomes, nonhomologous partner switches, and autosynapsis of X chromosome cores in meiotic spermatocytes. Fertility and meiosis are normal in Fkbp6 mutant females. Thus, Fkbp6 is a component of the synaptonemal complex essential for sex-specific fertility and for the fidelity of homologous chromosome pairing in meiosis.     

Protective Role of Comfrey Leave Extracts on UV-induced Zebrafish Fin Damage

In zebrafish, UV exposure leads to fin malformation phenotypes including fin reduction or absence. The present study evaluated UV-protective activities of comfrey leaves extracts in a zebrafish model by recording fin morphological changes. Chemopreventive effects of comfrey leave extracts were evaluated using Kaplan-Meier analysis and Cox proportional hazards regression. The results showed that (1) the mean times of return to normal fin in the UV+comfrey (50 and 100 ppm) groups were 3.43 and 2.86 days and were quicker compared with that in the UV only group (4.21 days); (2) zebrafish fins in the UV+comfrey (50 and 100 ppm) groups were 2.05 and 3.25 times more likely to return to normal than those in the UV only group; and (3) comfrey leave extracts had UV-absorbance abilities and significantly reduced ROS production in UV-exposed zebrafish embryos, which may attenuate UV-mediated apoptosis. In conclusion, comfrey leaves extracts may have the potential to be developed as UV-protective agents to protect zebrafish embryos from UV-induced damage.     

Bath immersion pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus)

Drug administration by immersion can be a preferable method in certain conditions especially for treating small-sized, anorexic, or valuable fish. Pharmacokinetic information regarding bath treatment is considerably lacking in comparison to other common administration routes. The current study aimed to investigate if immersion can be an effective route to administer florfenicol (FF) for treatment in Nile tilapia. Nile tilapia reared at 28°C were immersed with FF solution at concentrations of 50, 100, 200, 500, and 500/200 (3 hr/117 hr) ppm for 120 hr and moved to drug-free freshwater for another 24 hr. The serum FF concentration in 100, 200, and 500/200 ppm groups reached steady-state at 12 hr with concentrations of 2.44, 3.04, and 5.26 µg/ml, respectively, which were about 2% of the bathing concentrations. The target therapeutic levels of 1–4 µg/ml were attained and maintained within 1–12 hr, depending on the immersion concentration and the target MIC. Serum FF reached the target with shorter time at higher bathing concentration. Following the 120-hr bath, the serum FF declined with the first-order half-life of approximately 10 hr. A minimum of 100 ppm FF is required for treatment purpose, and an initial high loading concentration followed by maintenance concentration is a plausible way to reach in vivo therapeutic level in short time. Greater than 99% of the residual FF in the bathing water could be removed within 15 min by 0.05% NaOCl. Our results indicated that bath immersion is a promising potential route for FF administration in Nile tilapia.     

Antagonism of adenosine receptors by caffeine and caffeine metabolites in equine forebrain tissues

OBJECTIVE: To determine the presence of adenosine receptor subtypes A1 and A2a in equine forebrain tissues and to characterize the interactions of caffeine and its metabolites with adenosine receptors in the CNS of horses. SAMPLE POPULATION: Brain tissue specimens obtained during necropsy from 5 adult male research horses. PROCEDURE: Membrane-enriched homogenates from cerebral cortex and striatum were evaluated by radioligand binding assays with the A1-selective ligand [3H]DPCPX and the A2a-selective ligand [3H]ZM241385. Functional responses to adenosine receptor agonists and antagonists were determined by a nucleotide exchange assay using [35S]-guanosine 5'-(gamma-thio) triphosphate ([35S]GTPgammaS). RESULTS: Saturable high affinity [3H]DPCPX binding (A1) sites were detected in cerebral cortex and striatum, whereas high-affinity [3H]ZM241385 binding (A2a) sites were detected only in striatum. Caffeine and related methylxanthines had similar binding affinities at A1 and A2a sites with rank orders of drug binding affinities (theophylline > paraxanthine > or = caffeine > theobromine) similar to other species. [35S]GTPgammaS exchange revealed that caffeine and its metabolites act as pure adenosine receptor antagonists at concentrations that correspond to A1 and A2a receptor binding affinities. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study affirm the presence of guanine nucleotide binding protein linked adenosine receptors (ie, high-affinity A1 and A2a adenosine receptors) in equine forebrain tissues and reveal the antagonistic actions by caffeine and several biologically active caffeine metabolites. Antagonism of adenosine actions in the equine CNS by these stimulants may be responsible for some central actions of methylxanthine drugs, including motor stimulation and enhanced racing performance.     

Effect of sulfadiazine and pyrimethamine on selected physiologic and performance parameters in athletically conditioned thoroughbred horses during an incremental exercise stress test

Following the regimen used to treat equine protozoal myeloencephalitis, sulfadiazine (20 mg/kg) and pyrimethamine (1mg/kg) were administered orally once daily to 12 physically conditioned Thoroughbred horses for 4 consecutive days. The horses were randomly assigned to two test groups in a crossover design, with each horse serving as its own control. A stepwise exercise stress test was conducted to exhaustion. No effect on athletic performance was observed, and only marginal effects were noted in some hematologic and serochemical measurements, including decreased total white blood cell counts, red blood cell distribution width, total hemoglobin, serum sodium, and serum chloride. Serum folic acid concentration decreased significantly following sulfadiazine/pyrimethamine treatment.     

Effect of flunixin meglumine on selected physiologic and performance parameters of athletically conditioned thoroughbred horses subjected to an incremental exercise stress test

Twelve clinically sound, healthy, athletically conditioned Thoroughbred horses were subjected to an incremental exercise stress test to determine the effects and period of detection of a single dose of flunixin meglumine (1.1 mg/kg by intravenous injection) in serum and urine by ELISA. Flunixin concentrations, performance, and hematologic and clinical chemical parameters were measured. All horses were rotated through four treatment groups of a Latin-square design providing for each horse to serve as its own control. Flunixin meglumine reduced prostaglandin F(1alpha) and thromboxane concentrations that had been increased by intense exercise. Performance parameters did not improve and prostaglandin concentrations did not significantly correlate with total run time. Exercise did not change the flunixin elimination profile in either serum or urine, and concentrations were found to be below the detection limit of the ELISA test within 36 hours in serum and 120 hours in urine.     

Pharmacokinetics,optimal dosages and withdrawal time of florfenicol in Asian seabass (Lates calcarifer) after oral administration via medicated feed

Asian seabass (Lates calcarifer) is an economically important fish in Asian and Australian markets, but few pharmacokinetic (PK) data of antimicrobial drugs in this species is available. The present study investigated the PK behaviour of florfenicol (FF) through medicated feed in Asian seabass cultured at 25°C. The serum and muscle/skin concentrations of FF and its metabolite florfenicol amine (FFA) were determined by the HPLC-FLD method and analysed by one-compartmental model. The optimal dosages were determined by pharmacokinetic-pharmacodynamic (PK-PD) approach and the linear regression analysis was used to determine the withdrawal time (WDT). The PK study following a single oral administration of 15 mg/kg FF via medicated feed revealed that the absorption half-life (t_1/2Ka), elimination half-life (t_1/2K), peak concentration (C_max), area under the concentration-time curve (AUC), volume of distribution (Vd/F) and clearance (CL/F) were 1.47 h, 8.07 h, 8.61 μg/ml, 146.41 h·μg/ml, 1.19 L/kg and 0.102 L/kg/h, respectively. The muscle/skin concentration-time profile was similar to that of the serum, suggesting well distribution but only a small fraction of FF was metabolized to FFA. The optimal dosage for a minimum inhibitory concentration of 2 μg/ml was calculated as 13.38 mg/kg/day. The appropriate WDT after multiple oral medications with 15 mg/kg FF once daily for 7 days was determined as 8 days. Information obtained from the current study can potentially be applied for the treatment of bacterial diseases in farming Asian seabass.     

Method for bacterial expression and purification of sesame cystatin via artificial oil bodies

A method was developed for production of sesame cystatin, a thermostable cysteine protease inhibitor. Sesame cystatin was first expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a short hydrophilic linker peptide. Stable artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble oleosin-cystatin fusion protein. After centrifugation, the oleosin-cystatin fusion protein was exclusively found in the artificial oil bodies. Proteolytic cleavage with papain, a cysteine protease effectively inhibited by cystatin, separated soluble cystatin from oleosin that was firmly embedded in the artificial oil bodies. After recentrifugation, papain that coexisted with cystatin in the collected supernatant was denatured by incubating at 55 degrees C for 30 min. The insoluble denatured papain was removed by one more centrifugation, and the expressed cystatin of high yield and purity was harvested simply by concentrating the ultimate supernatant. Comparable inhibitory activity toward papain was observed between the expressed cystatin and the native one purified from sesame seeds. This method is presumably applicable to production of other protease inhibitors whose target proteases are economically available.