Stability studies of the metabolites of nitrofuran antibiotics during storage and cooking

Nitrofuran antibiotics cannot be used in food production within the European Union because of their potential health risks to consumers. The recent discovery of their widespread use in global food industries and the finding of semicarbazide in baby food as a result of packaging contamination have focused attention on the toxicity and stability of these drugs and their metabolites. The stability of the nitrofuran marker residues 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) were tested. Muscle and liver of nitrofuran treated pigs were cooked by frying, grilling, roasting and microwaving. Between 67 and 100% of the residues remained after cooking, demonstrating that these metabolites are largely resistant to conventional cooking techniques and will continue to pose a health risk. The concentration of metabolites in pig muscle and liver did not drop significantly during 8 months of storage at -20 degrees C. Metabolite stock and working standard solutions in methanol were also stable for 10 months at 4 degrees C. Only a 10 ng ml(-1) solution of SEM showed a small drop in concentration over this extended storage period.

Na+/H+ exchange inhibitor cariporide attenuates skeletal muscle infarction when administered before ischemia or reperfusion

Administration of Na(+)/H(+) exchange isoform-1 (NHE-1) inhibitors before ischemia has been shown to attenuate myocardial infarction in several animal models of ischemia-reperfusion injury. However, controversy still exists as to the efficacy of NHE-1 inhibitors in protection of myocardial infarction when administered at the onset of reperfusion. Furthermore, the efficacy of NHE-1 inhibition in protection of skeletal muscle from infarction (necrosis) has not been studied. This information has potential clinical applications in prevention or salvage of skeletal muscle from ischemia-reperfusion injury in elective and trauma reconstructive surgery. The objective of this research project is to test our hypothesis that the NHE-1 inhibitor cariporide is effective in protection of skeletal muscle from infarction when administered at the onset of sustained ischemia or reperfusion and to study the mechanism of action of cariporide. In our studies, we observed that intravenous administration of cariporide 10 min before ischemia (1 or 3 mg/kg) or reperfusion (3 mg/kg) significantly reduced infarction in pig latissimus dorsi muscle flaps compared with the control, when these muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion (P <0.05; n = 5 pigs/group). Both preischemic and postischemic cariporide treatment (3 mg/kg) induced a significant decrease in muscle myeloperoxidase activity and mitochondrial-free Ca(2+) content and a significant increase in muscle ATP content within 2 h of reperfusion (P <0.05; n = 4 pigs/group). Preischemic and postischemic cariporide treatment (3 mg/kg) also significantly inhibited muscle NHE-1 protein expression within 2 h of reperfusion after 4 h of ischemia, compared with the control (P <0.05; n = 3 pigs/group). These observations support our hypothesis that cariporide attenuates skeletal muscle infarction when administered at the onset of ischemia or reperfusion, and the mechanism involves attenuation of neutrophil accumulation and mitochondrial-free Ca(2+) overload and preservation of ATP synthesis in the early stage of reperfusion.

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury:efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning:efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P <0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P <0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P <0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P <0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.

Mammalian triokinase and dihydroxyacetone kinase are the same enzyme

It is widely accepted that the ATP-dependent phosphorylation of D-glyceraldehyde in the fructokinase pathway of fructose metabolism requires the enzyme “triokinase”. However, experimental data on this enzyme are remarkably scarce. The enzyme has been purified from a variety of sources and peptides derived from the pig kidney enzyme show high similarity to human dihydroxyacetone kinase – an enzyme which also has FMN cyclase activity in high manganese ion concentrations. The properties of the two enzymes are also highly similar. Therefore it is proposed that mammalian triokinase and dihydroxyacetone kinase are, in fact, the same enzyme. This has consequences for investigations of normal and aberrant fructose metabolism and for the teaching of biochemistry in medical and science courses.

Identification of serum stress biomarkers in pigs housed at different stocking densities

Eight Duroc × (Landrace × Large White) male pigs housed at a stocking rate of 0.50 m2/pig were subjected to a higher stocking rate of 0.25 m2/pig (higher density, HD) for two 4-day periods over 26 days. Using biochemical and proteomic techniques serum and plasma samples were examined to identify potential biomarkers for monitoring stress due to HD housing. HD housed pigs showed significant differences (P < 0.001) in total cholesterol and low density lipoprotein-associated cholesterol, as well as in concentrations of the pig-major acute phase protein (Pig-MAP) (P = 0.002). No differences were observed in serum cortisol or other acute phase proteins such as haptoglobin, C-reactive protein or apolipoprotein A–I. HD-individuals also showed an imbalance in redox homeostasis, detected as an increase in the level of oxidized proteins measured as the total plasma carbonyl protein content (P < 0.001) with a compensatory increase in the activity of the antioxidant enzyme glutathione peroxidase (P = 0.012). Comparison of the serum proteome yielded a new potential stress biomarker, identified as actin by mass spectrometry. Cluster analysis of the results indicated that individuals segregated into two groups, with different response patterns, suggesting that the stress response depended on individual susceptibility.

Proteomics on porcine haptoglobin and IgG/IgA show protein species distribution and glycosylation pattern to remain similar in PCV2-SD infection

Haptoglobin (Hp) and immunoglobulins are plasma glycoproteins involved in the immune reaction of the organism after infection and/or inflammation. Porcine circovirus type 2-systemic disease (PCV2-SD), formerly known as postweaning multisystemic wasting syndrome (PMWS), is a globally spread pig disease of great economic impact. PCV2-SD affects the immunological system of pigs causing immunosuppression. The aim of this work was to characterize the Hp protein species of healthy and PCV2-SD affected pigs, as well as the protein backbone and the glycan chain composition of porcine Hp. PCV2-SD affected pigs had an increased overall Hp level, but it did not affect the ratio between Hp species. Glycoproteomic analysis of the Hp β subunits confirmed that porcine Hp is N-glycosylated and, unexpectedly, O-glycosylated, a PTM that is not found on Hp from healthy humans. The glyco-profile of porcine IgG and IgA heavy chains was also characterized; decreased levels of both proteins were found in the investigated group of PCV2-SD affected pigs. Obtained results indicate that no significant changes in the N- and O-glycosylation patterns of these major porcine plasma glycoproteins were detectable between healthy and PCV2-SD affected animals.

Deoxynivalenol exposure assessment in young children in Tanzania

Scope: This study assessed deoxynivalenol (DON) exposure in children from three geographic locations within Tanzania, over three time points in 1 year, using a urinary biomarker of exposure.

Methods and results: A total of 166 children aged 6-14 months were studied at a maize harvest and followed up twice at 6-month intervals. On two consecutive days, morning urine was collected from each child and urinary DON was measured using an LC-MS method, with and without beta-glucuronidase hydrolysis in order to assess free DON (fDON) and glucuronide DON. Overall, urinary DON increased significantly along with the three visits (geometric mean 1.1, 2.3, and 5.7 ng/mL, at visits 1, 2, and 3, respectively, p <0.01). fDON was 22% of urinary total DON. Urinary DON excretion rate was 74% in village Kikelelwa based on food DON level and food consumption. Assuming 360 mL of urine excreted per day, 10, 19, and 29% of children at visits 1, 2, and 3, respectively, exceeded the provisional maximum tolerable daily intake of 1000 ng/kg b.w./day.

Conclusion: Young children in Tanzania are chronically exposed to DON due to eating contaminated maize, although exposure levels varied markedly by region and season.

Lack of in vitro-in vivo correlation for a UC781-releasing vaginal ring in macaques

This study describes the preclinical development of a matrix-type silicone elastomer vaginal ring device designed to provide controlled release of UC781, a non-nucleoside re- verse transcriptase inhibitor. Testing of both human- and macaque-sized rings in a sink condition in vitro release model demonstrated continuous UC781 release in quantities consid- ered sufficient to maintain vaginal fluid concentrations at levels 82–860-fold higher than the in vitro IC50 (2.0 to 10.4 nM) and therefore potentially protect against mucosal trans- mission of HIV. The 100-mg UC781 rings were well tolerated in pig-tailed macaques, did not induce local inflammation as determined by cytokine analysis and maintained median con- centrations in vaginal fluids of UC781 in the range of 0.27 to 5.18 mM during the course of the 28-day study. Analysis of residual UC781 content in rings after completion of both the in vitro release and macaque pharmacokinetic studies revealed that 57 and 5 mg of UC781 was released, respectively. The pharmacokinetic analysis of a 100-mg UC781 vaginal ring in pig-tailed macaques showed poor in vivo–in vitro correlation, attributed to the very poor solubility of UC781 in vaginal fluid and resulting in a dissolution-controlled drug release mecha- nism rather than the expected diffusion-controlled mechanism.

T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37 degreesC using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs+-rich pipette solutions to block K+ currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V-1/2 of -45 +/- 5 mV. In contrast, the T current inactivated with a V-1/2 of -80 +/- 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 +/- 54 nm) and Ni2+ (ED50 = 65 +/- 16 muM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by TTX, reduction in external Na+, application of nifedipine at concentrations below 300 nm or substitution of external Ca2+ with Ba2+, but was reduced by Ni2+ with an ED50 of 6 +/- 1 mum. When cells were stepped from -100 to -30 mV in Ca2+-free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg2+ with an ED50 of 32 +/- 16 mum. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication. (C) 2003 Elsevier B.V. All rights reserved.

Docking the value of pigmeat? Prevalence and financial implications of welfare lesions in Irish slaughter pigs.

Expansion of the meat inspection process to incorporate animal-based welfare measurements could contribute towards significant improvements in pig (Sus scrofa domesticus) welfare and farm profitability. This study aimed to determine the prevalence of different welfare-related lesions on the carcase and their relationship with carcase condemnations (CC) and carcase weight (CW). The financial implications of losses associated with CC and CW reductions related to the welfare lesions were also estimated. Data on tail lesions, loin bruising and bursitis, CW and condemnation/trimming outcome (and associated weights) were collected for 3,537slaughter pigs (mean [± SEM] carcase weight: 79.2 [± 8.82] kg). Overall, 72.5% of pigs had detectable tail lesions, whilst 16.0 and 44.0% were affected by severe loin bruising and hind limb bursitis, respectively. There were 2.5% of study carcases condemned and a further 3.3% were trimmed. The primary cause of CC was abscessation. While tail lesion severity did not increase the risk of abscessation, it was significantly associated with CC. Male pigs had a higher risk of tail lesions and of CC. The financial loss to producers associated with CC and trimmings was estimated at €1.10 per study pig. CW was reduced by up to 12 kg in cases of severe tail lesions. However, even mild lesions were associated with a significant reduction in CW of 1.2 kg. The value of the loss in potential CW associated with tail lesions was €0.59 per study pig. Combined with losses attributable to CC and trimmings this represented a loss of 43% of the profit margin per pig, at the time of the study, attributable to tail biting. These findings illustrate the magnitude of the impact of tail biting on pig welfare and on profitability of the pig industry. They also emphasise the potential contribution that the inclusion of welfare parameters at meat inspection could make to pig producers in informing herd health and welfare management plans.

Phylogeny and ancient DNA of Sus provides insights into neolithic expansion in Island Southeast Asia and Oceania

Human settlement of Oceania marked the culmination of a global colonization process that began when humans first left Africa at least 90,000 years ago. The precise origins and dispersal routes of the Austronesian peoples and the associated Lapita culture remain contentious, and numerous disparate models of dispersal (based primarily on linguistic, genetic, and archeological data) have been proposed. Here, through the use of mtDNA from 781 modern and ancient Sus specimens, we provide evidence for an early human-mediated translocation of the Sulawesi warty pig (Sus celebensis) to Flores and Timor and two later separate human-mediated dispersals of domestic pig (Sus scrofa) through Island Southeast Asia into Oceania. Of the later dispersal routes, one is unequivocally associated with the Neolithic (Lapita) and later Polynesian migrations and links modern and archeological Javan, Sumatran, Wallacean, and Oceanic pigs with mainland Southeast Asian S. scrofa. Archeological and genetic evidence shows these pigs were certainly introduced to islands east of the Wallace Line, including New Guinea, and that so-called "wild" pigs within this region are most likely feral descendants of domestic pigs introduced by early agriculturalists. The other later pig dispersal links mainland East Asian pigs to western Micronesia, Taiwan, and the Philippines. These results provide important data with which to test current models for human dispersal in the region. © 2007 by The National Academy of Sciences of the USA.

The ones that come ready made:The identification and use of Sus tusks as tools at prehistoric cave sites in Malaysia

Evidence for osseous technologies has featured in excavation reports from Southeast Asia for almost a century and from archaeological deposits as old as 43,000 years BP. However, in contrast to the significance that is placed on this technology in other parts of the world, until recently, Southeast Asian assemblages have drawn only very limited attention. Concentrating on evidence from Malaysia, the current paper examines one element of this inventory of tools: the deliberate modification of pig canines and the means by which such alteration can be distinguished from patterns of natural tooth wear. Particular attention is paid to the bearded pig (Sus barbatus), as it is one of the two species of wild boar in Malaysia whose tusks are most likely to have been used by prehistoric toolmakers. Reference is also made to wider, regional ethnographic examples of known tusk implements and their accredited uses to further assist in the identification process. Distinguishing criteria for worked tusk are formulated according to the type and extent of modification. These criteria are then applied to archaeological specimens recovered from two prehistoric cave sites in Malaysia, Gua Bintong and Niah Cave.

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: a novel CRF-like peptide with smooth muscle contraction activity

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations.