Nickel and copper residues in meat from wild artiodactyls hunted with nickel-plated non-lead rifle bullets

A nickel (Ni)-plated copper-solid bullet type released up to 93 μg Ni /10 g bullet mass when immersed into meat juice for 7 days (to simulate fragments remaining in venison). A non-nickel-plated counterpart of identical construction released no Ni, but up to 250 μg copper. During thermal processing of pork cubes with embedded bullets, an average of 2.8 and up to 4.3 (maximum) μg Ni were released from the Ni-plated bullet to the surrounding meat. Average nickel and copper content in meat samples (taken in 2–3 cm distance from the shot wounds) from 30 roe deer (Capreolus capreolus) and 3 sika deer (Cervus nippon) killed with nickel-plated copper bullets did not differ significantly from those in controls (roe deer haunch). Contamination scenarios would Cu and Ni contents per portion increase moderately by 20 and 3.3 μg, respectively. In order to limit alimentary Ni uptake, the technological need for Ni-plating of bullets should be carefully evaluated.     

Microbiological condition and shelf life of meat from hunted game birds

Hunted game birds (eight partridges, nine wood pigeons, 25 quails, 16 chilled and 16 frozen–thawed pheasants) were processed according to “Good Manufacturing Practice” rules. Microflora of skin, intestinal content and meat cuts (breast and thigh, both fresh and stored in vacuum package) was analysed. Listeria monocytogenes, Salmonella sp. and Campylobacter sp. were not recovered from any sample. Log microbial numbers on skin or in intestines were not significantly related to those on meat cuts. With the exception of pigeons, microbial numbers of the two meat cuts did not differ significantly (p > 0.05), and no significant increase in microbial numbers in vacuum-stored meat was found; the same applied to frozen–thawed compared to chilled pheasants. On meat, average total viable counts were <4.00 log cfu/cm² with a maximum of 6.48 log cfu/cm². Median Escherichia coli numbers were <2.00 log cfu/cm², maximum was 4.48 log cfu/cm². Meat cuts obtained from partridges, quails and pheasants demonstrated a shelf life of 1 week, provided they were kept vacuum-packaged at 0°C to 1°C.     

Blood sampling by puncture in the cavernous sinus from hunted wild boar

A new method of sampling based on the extraction of blood from the cavernous sinus of the dura mater has been assessed in hunted wild boar. Blood from 139 animals was obtained by two different extraction methods: the harvesting from thoracic cavity (TC) and intracavernous venipuncture (IV). Sera obtained by the IV method had higher volume (mean 2.85 vs 1.85 ml), were less hemolytic (mean absorbance at 450 nm: 1.01 vs 2.41 nm). A higher number of samples and a higher proportion of sera collected by IV (90.6 %) compared to those obtained using the TC method (78.4 %), could be analyzed against Aujeszky’s disease using blocking ELISA. No statistically significant differences in seroprevalences between samples obtained using both extraction methods were observed. The results obtained indicate that the IV is an easy, fast, reliable, clean, and safe method to collect blood samples from hunted wild boar, proving a real alternative to the traditional collection method.     

Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae. Kinetics and mechanism

The mechanism of copper uptake in Saccharomyces cerevisiae has been investigated using a combination of 64Cu2+ and atomic absorption spectrophotometry. A wild type copper-resistant CUP 1R-containing strain and a strain carrying a deletion of the CUP1 locus (yeast copper metallothionein) exhibited quantitatively similar saturable energy-dependent 64Cu2+ uptake when cultures were pregrown in copper-free media (medium [Cu] approximately 15 nM). The kinetic constants for uptake by the wild type strain were Vmax = 0.21 nmol of copper/min/mg of protein and Km = 4.4 microM. This accumulation of 64Cu2+ represented net uptake as confirmed by atomic absorption spectrophotometry. This uptake was not seen in glucose-starved cells, but was supported in glycerol- and ethanol-grown ones. Uptake was inhibited by both N3- and dinitrophenol and was barely detectable in cultures at 4 degrees C. When present at 50 microM, Zn2+ and Ni2+ inhibited by 50% indicating that this uptake process was relatively selective for Cu2+. 64Cu2+ accumulation was qualitatively and quantitatively different in cultures either grown in or preincubated with cold Cu2+. Either treatment resulted in the appearance of a fast phase (t 1/2 approximately 1 min) of 64Cu2+ accumulation which represented isotopic exchange since it did not lead to an increase in the mass of cell-associated copper; also, it was not energy-dependent. Exchange of 64Cu2+ into this pool was not inhibited by Zn2+. Pretreatment with Cu2+ caused a change in the rate of net accumulation as well; a 3-h incubation of cells in 5 microM medium Cu2+ caused a 1.6-fold increase in the velocity of energy-dependent uptake. Prior addition of cycloheximide abolished this Cu2(+)-dependent increase and, in fact, inhibited the 64Cu2+ uptake velocity by greater than 85%. The exchangeable pool was also absent in cycloheximide, Cu2(+)-treated cells suggesting that exchangeable Cu2+ derived from the copper taken up initially by the energy-dependent process. The thionein deletion mutant was similar to wild type in response to medium Cu2+ and cycloheximide indicating that copper metallothionein is not directly involved in Cu2+ uptake (as distinct from retention) in yeast.     

Mutagenicity of pan residues and gravy from fried meat

Lean pork meat was fried with or without the addition of frying-fat at 200 or 250 degrees C. The pan residues were collected by washing the hot pan with boiling water. When producing thickened gravy the water was substituted by a mixture of water and flour, milk and flour or cream and flour. The basic extracts were tested for mutagenicity in Ames' Salmonella test on strain TA98 with the addition of S9 mix. High amounts of mutagenicity were found in all samples. The amounts of mutagenicity in the pan residues were at a comparable level of the amounts found in the meat crusts. Thickening of the gravy caused only small changes in the mutagenicity.     

Investigating the risks of removing wild meat from global food systems

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Higher plants possess two different types of ATX1-like copper chaperones

Copper (Cu) chaperones constitute a family of small Cu+-binding proteins required for Cu homeostasis in eukaryotes. The ATX1 family of Cu chaperones specifically delivers Cu to heavy metal P-type ATPases. The plant Arabidopsis thaliana expresses the ATX1-like Cu chaperone CCH, which exhibits a plant-specific carboxy-terminal domain (CTD) with unique structural properties. We show that CCH homologues from other higher plants contain CTDs with structural properties similar to Arabidopsis CCH. Furthermore, we identify a new ATX1-like Cu chaperone in Arabidopsis, AtATX1, which functionally complements yeast atx1Delta and sod1Delta associated phenotypes, and localizes to the cytosol of Arabidopsis cells. Interestingly, AtATX1, but not full-length CCH, interacts in vivo with the Arabidopsis RAN1 Cu-transporting P-type ATPase by yeast two-hybrid. We propose that higher plants express two types of ATX1-like Cu chaperones: the ATX1-type with a predominant function in Cu delivery to P-type ATPases, and the CCH-type with additional CTD-mediated plant-specific functions.     

Drought responses in Aleppo pine seedlings from two wild provenances with different climatic features

Global warming will likely exacerbate the negative effects of limited water availability in the Mediterranean area. The Italian Aleppo pine (Pinus halepensis Mill.) provenances are distributed along the coasts except Otricoli provenance growing in an unusual location between 300 and 1,000 m a. s. l., in Umbria (central Italy). The aim of the present study was to investigate the photosynthetic response to a 28-day-long drought and to a subsequent reestablishment of water availability in Otricoli and North Euboea (Greece) provenances, representing different locations along a rainfall gradient in the natural range of this species. Six-month-old seedlings were used in this experiment since at this age Aleppo pine plants in Mediterranean climate face their first water stress potentially affecting plant survival. Water potential (ψ_w), net photosynthesis and stomatal conductance decreased during drought in both provenances and showed minimal values 28 days after beginning the treatment (DAT). Otricoli seedlings adjusted ψ_w gradually as the stress level increased and 21 DAT showed a lower ψ_w than North Euboea. In contrast, in North Euboea seedlings ψ_w that was not affected until 21 DAT rapidly dropped to a minimum of −3.81 MPa 28 DAT. At the onset of the stress the intercellular CO₂ concentration (C _i) was reduced, and the “instrinsic” water-use efficiency (WUE_i) was enhanced in both provenances, as stomatal conductance decreased more rapidly than photosynthesis. However, 28 DAT, C _i increased and WUEi decreased as stomatal conductance and photosynthesis declined to minimum levels, revealing nonstomatal limitations of photosynthesis. A rapid decrease in PSII maximal photochemical efficiency estimated by chlorophyll (Chl) fluorescence (F_v/F_m) was also observed when the stress became severe. At the final stage of water stress, North Euboea seedlings maintained significantly higher values of F_v/F_m than Otricoli seedlings. Upon rewatering, photosynthesis did not fully recover in Otricoli seedlings (41 DAT), while all other parameters recovered to control levels in both provenances. No drought-induced physiological differences were consistent with the regional climatic features of these two provenances. Our results suggest that phenotypic plasticity in drought response may help Otricoli provenance cope with global warming, but that recurrent drought episode may slow down the primary productivity of this provenance.     

Copper toxicity in Alzheimer's disease: cognitive loss from ingestion of inorganic copper

In this review I present the hypothesis that a toxic substance, inorganic copper, ingested from drinking water and vitamin/mineral supplements containing inorganic copper, is at least partially causal of the epidemic of Alzheimer's disease (AD) we are seeing in developed countries. I set the stage for this hypothesis by pointing out that the epidemic is a new disease phenomenon coinciding temporally with the use of copper plumbing in developed countries. The evidence is good that AD was nonexistent or rare in the 1800 s and early 1900 s, and the arguments that elderly people did not exist in those times, or that AD was simply attributed to senility, are refuted. The web of evidence tying ingestion of inorganic copper as a causal factor in AD is strong, and includes AD animal model data where trace amounts of inorganic copper in the drinking water markedly worsened AD, human studies where ingestion of copper supplements, along with a high fat diet, is associated with a marked loss of cognition, human studies showing a markedly higher mortality in elderly women ingesting copper supplements, as well as other data. It is likely that a high fat diet works in conjunction with ingestion of inorganic copper to increase the risk of AD. It is clear that some factor toxic to the brain is present in the environment in developed countries, but not undeveloped countries, and is a major risk factor for AD. I believe that that toxic factor is ingestion of inorganic copper.     

Identification of active-site histidine residues of a self-incompatibility ribonuclease from a wild tomato.

The style component of the self-incompatibility (S) locus of the wild tomato Lycopersicon peruvianum (L.) Mill. is an allelic series of glycoproteins with ribonuclease activity (S-RNases). Treatment of the S3-RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of RNase activity. In the presence of a competitive inhibitor, guanosine 3'-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetate was reduced significantly. Analysis of the tryptic digestion products of the iodoacetate-modified S-RNase by reversed-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence of 3'-GMP. Histidine-88 was also modified, but this occurred both in the presence and absence of 3'-GMP, suggesting that this residue is accessible when 3'-GMP is in the active site. Cysteine-150 was modified by iodoacetate in the absence of 3'-GMP and, to a lesser extent, in its presence. The results are discussed with respect to the related fungal RNase T2 family and the mechanism of S-RNase action.     

Beta-2 microglobulin types in mice of wild origin

To determine the distribution of beta-2 microglobulin (B2m) alleles in wild mice we have typed mice derived from natural populations in Europe, North Africa, South America, and East Asia. Mus musculus domesticus mice from Germany, France, Italy, and Peru were all B2m ^a as were most from the United Kingdom. M.m. musculus mice from Denmark and Czechoslovakia, several stocks of M.m. molossinus from Japan, and M.m. castaneus from China, Thailand, and the Philippines were of B2m ^b type. This is consistent with the notion that C57BL/6 may have obtained some of its genes, including B2m, from Eastern mice. A BgII restriction site characteristic of B2m ^b was also found in mice from Czechoslovakia and Japan, confirming that B2m ^b is a naturally occurring allele of B2m. A new type of ₂m ( ₂m^w1) was found in four stocks of M. spretus from Portugal, Spain, and Morocco. This molecule differs in apparent size and charge from the a and b types. ₂m^w2 was found together with ₂ ma in one stock of M.m. domesticus (brevirostris) from Morocco. ₂m^w3 and ₂m^w4 were found in a few M. m. bactrianus from Pakistan. In all cases tested, these new ₂m molecules associate with class I histocompatibility antigens.Abbreviations used in this paper ₂m beta-2 microglobulin - B2m gene for beta-2 microglobulin - IEF isoelectric focusing - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - MHC major histocompatibility complex - T. E. Tris-EDTA buffer     

Heritable morphological differentiation in salmonids from two distinct stream types

We tested for phenotype-to-habitat associations in brown trout Salmo trutta populations from two ecologically different habitat types; i.e., groundwater and surface-water-fed streams. Additionally, we raised captive offspring from two such populations under standardised conditions to test whether potential phenotypic differentiation would be passed on to offspring. We found analogous differentiation by habitat in multiple wild populations. Some of these morphological differences were at least partially inherited by offspring. We suggest that this could have implications for both scientists and fisheries authorities studying or managing trout populations.     

Control of transmission with two types of infection

The effectiveness of a vaccination strategy to control transmission of an infectious disease depends on the way vaccine doses are distributed to individuals in a community of households. Here we show that this dependence is more complicated when acquisition and severity of illness are determined by the size of the infecting dose, as is thought to be the case for measles and varicella. Two alternative formulations for the way vaccination changes an individual's susceptibility and infectivity show that vaccination coverage, the nature of the vaccine response and the distribution of household size also have a big impact on which strategy is more effective. These judgements are made by comparing the post-vaccination reproduction numbers corresponding to different vaccination strategies.     

Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C(18) bonded silica column with a deionized water-methanol-acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/kg were in the range of 71.4-99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/kg were in the range of 6.2-13.9% and 4.0-8.7%, respectively. The intra-day precision (n=5) for nitroimidazoles residues in chicken spiked at 20 microg/kg is 6.9%, and the inter-day precision for 5 days (n=25) is 11%. The method is capable of identifying nitroimidazole residues at > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.     

Identification of modified tryptophan residues in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein

Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu(2+), treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu(2+)-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu(2+)-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu(2+)-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu(2+)-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.     

Structure of actin-containing filaments from two types of non-muscle cells

Bundles of actin-containing filaments from the acrosomal process of horseshoe crab sperm and from sea urchin egg contain a second protein having a molecular weight of about 55,000. Electron micrographs of these filamentous bundles show features reminiscent of paracrystalline arrays of actin except that bundles from the sea urchin egg have distinctive transverse bands every 110 Å. From optical diffraction patterns of the micrographs, we deduced very similar models for both structures. The models consist of hexagonal arrays of actin filaments cross-linked by the second protein. The pattern of transverse bands in bundles derived from the sea urchin eggs is accounted for by postulating that the second protein is bonded to actin only at positions where cross-linking can occur, rather than being bonded to every actin. The helical symmetry of the actin requires that the bonding contacts involved in the cross-linking be slightly different at different positions along the length of the bundle. The technique of image reconstruction was used to obtain a three-dimensional map of the bundles from the acrosomal process.     

Cryo-electron microscopy reconstructions of two types of wild rabbit hemorrhagic disease viruses characterized the structural features of Lagovirus

Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection. The etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the Caliciviridae family. Compared to other calicivirus, such as rNV and SMSV, the structure of Lagovirus members is not well characterized. In this report, structures of two types of wild RHDV particles, the intact virion and the core-like particle (CLP), were reconstructed by cryo-electron microscopy at 11Å and 17Å, respectively. This is the first time the 3D structure of wild caliciviruses CLP has been provided, and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus. Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated. In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus, the capsomers of RHDV virion exhibited unique structural features and assembly modes. Both P1 and P2 subdomains have interactions inside the AB capsomer, while only P2 subdomains have interaction inside CC capsomer. The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting, and the rotation of RHDV VP60 P domain with respect to its S domain, compared with SMSV, was observed. Collectively, our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus, which is important for functional analysis and better vaccine development in the future.