Copper physiology in ruminants: trafficking of systemic copper, adaptations to variation in nutritional supply and thiomolybdate challenge.

Ruminants are recognised to suffer from Cu-responsive disorders. Present understanding of Cu transport and metabolism is limited and inconsistent across vets and veterinary professionals. There has been much progress from the studies of the 1980s and early 1990s in cellular Cu transport and liver metabolism which has not been translated into agricultural practice. Cu metabolism operates in regulated pathways of Cu trafficking rather than in pools of Cu lability. Cu in the cell is chaperoned to enzyme production, retention within metallothionein or excretion via the Golgi into the blood. The hepatocyte differs in that Cu-containing caeruloplasmin can be synthesised to provide systemic Cu supply and excess Cu is excreted via bile. The aim of the present review is to improve understanding and highlight the relevant progress in relation to ruminants through the translation of newer findings from medicine and non-ruminant animal models into ruminants.

Devising a training programme and reviewing the lessons identified during contingency operations on board the Role 3 Maritime platform.

The deployment of the Primary Casualty Receiving Facility (PCRF) to Sierra Leone in October 2014 in support of Operation GRITROCK was a contingency operation that required a very specific training programme. This article discusses the training and how it evolved beyond its original remit into a programme used as a management tool to meet the needs of the staff. The result was the implementation of a comprehensive package from which lessons can be drawn and used when planning future operations.

A standardised protocol for the validation of banking methodologies for arterial allografts.

The objective of this study was to design and test a protocol for the validation of banking methodologies for arterial allografts. A series of in vitro biomechanical and biological assessments were derived, and applied to paired fresh and banked femoral arteries. The ultimate tensile stress and strain, suture pullout stress and strain, expansion/rupture under hydrostatic pressure, histological structure and biocompatibility properties of disinfected and cryopreserved femoral arteries were compared to those of fresh controls. No significant differences were detected in any of the test criteria. This validation protocol provides an effective means of testing and validating banking protocols for arterial allografts.

Tissue and corneal donation and transplantation in the UK.

NHS Blood and Transplant (NHSBT) was established in 2005 as a Special Health Authority when the National Blood Authority and UK Transplant merged. This helped to bring tissue banking and organ transplantation services under one umbrella organization. This merger means that ~!95% of all deceased donors (whether tissue, organ or both) are now facilitated by one organization. NHSBT Tissue Services is the largest tissue establishment in the UK, and is a multi-tissue bank that specializes in the consent, retrieval, processing, storage, and dispatch of donated tissue coordinated from a purpose built, state-of-the-art tissue bank in Liverpool. Tissue donations can come from either tissue-only donors or solid organ donors who also donate tissue. Annually there are ~450 multi-tissue donors and 2500 eye donors in the UK, resulting in many thousands of transplants, including 3564 cornea transplants in 2010-2011. The separation of tissue- and organ-specific donors is largely artificial, and while organ transplantation can be life-saving, tissue transplantation can also have a dramatic effect on a patient's quality of life. It is hoped that all donors, both organ and tissue, will be recognized for the gift they make to society after their death.

Comparison of X-ray absorption spectra from copper-loaded bovine and ovine livers.

BACKGROUND: Copper toxicity and hepatic copper accumulation pose a serious risk to ruminant health and production. Differences in the copper-handling mechanisms of cattle and sheep have been noted, not only in comparison to each other, but also in comparison to 'copper-tolerant' monogastric species. Ruminants appear less able to cope with rising liver copper concentration than monogastric counterparts, with sheep in general less able to cope with elevated copper intake than cattle. METHODS: X-ray absorption spectroscopy (XAS) was used to investigate the differences between the livers of these species at high copper status. RESULTS: The X-ray absorption fine structure (XAFS) and X-ray absorption near edge structure (XANES) spectra indicated that the hepatic copper compound is most likely to be bound to metallothionein; consistent with monogastric species. CONCLUSION: Although, most likely stored as copper-metallothionein, there may be a role for glutathione as a short-term, intermediate copper buffer which may have more relevance to sheep than cattle. The potential that thiomolybdate bound copper can be stored in the liver could not be ruled out.

UK ruminant farmer understanding of copper-related terminology.

Copper provision is not straightforward in ruminants. Other dietary elements such as iron and molybdenum in combination with sulphur are able to interfere with copper availability, absorption and function. These complexities surrounding copper availability in cattle and sheep prevent the simple calculation of copper requirements. Previous research has established that UK farms are failing to provide copper in an effective manner, with some over-supplying and others under-supplying. Copper terminology is not consistently understood by professionals in the agricultural industry; potentially resulting in confusing or misinformation being passed on to farmers. The present work found that most (84 %) farmers felt they lacked understanding of copper related problems and their associated terminology. However, farmers who felt they had the least knowledge appeared more likely to underestimate their knowledge, and those who felt more confident in their knowledge were more likely to be overestimating it. Simple over- and under-supply of copper were only recognised by a small proportion (17 %) as causes of copper related problems while more complex issues were better recognised (27 %) as potential causal factors. However, the more specific terminology relating to the molybdenum-sulphur antagonism was poorly understood by most respondents (96 %).

X-ray absorption spectroscopy of copper and iron in sheep digesta.

BACKGROUND: The bioavailable supply of copper to ruminants has long been problematic. Complexities in supply exist due to interactions with other dietary elements in the rumen, most notably with iron or molybdenum in combination with sulphur, which can result in copper binding preventing its absorption. The molybdenum-sulphur-copper interaction has been extensively studied over the years. However, very little is known about the iron-sulphur-copper interaction, especially its mode of action in the gastrointestinal tract. METHODS: In the present work digesta from the rumen and jejunum of sheep fed a high copper, sulphur and iron diet was analysed using X-ray absorption spectroscopy (XAS). RESULTS: X-ray absorption fine structure (XAFS) and X-ray absorption near edge structure (XANES) indicated that all of the copper and iron had changed in bonding in the rumen and that the oxidation state of the elements had been reduced into a mix of Fe2+ & Fe3+ and Cu+ with some Cu0. CONCLUSION: The copper compounds were most likely to be thiol co-ordinated in line with Cu+ chemistry. Changes to the copper compounds took place in the jejunum, although thiols were still highly favoured the possible existence of a copper-iron-sulphur complex which also included oxygen and chloride was also observed. This possibly has some resemblance to the crystal structure of bornite.

A combination of serum leucine-rich α-2-glycoprotein 1, CA19-9 and interleukin-6 differentiate biliary tract cancer from benign biliary strictures.

BACKGROUND: Biliary tract cancer (BTC) and benign biliary strictures can be difficult to differentiate using standard tumour markers such as serum carbohydrate antigen 19-9 (CA19-9) as they lack diagnostic accuracy. METHODS: Two-dimensional difference gel electrophoresis and tandem mass spectrometry were used to profile immunodepleted serum samples collected from cases of BTC, primary sclerosing cholangitis (PSC), immunoglobulin G4-associated cholangitis and healthy volunteers. The serum levels of one candidate protein, leucine-rich α-2-glycoprotein (LRG1), were verified in individual samples using enzyme-linked immunosorbent assay and compared with serum levels of CA19-9, bilirubin, interleukin-6 (IL-6) and other inflammatory markers. RESULTS: We report increased LRG1, CA19-9 and IL-6 levels in serum from patients with BTC compared with benign disease and healthy controls. Immunohistochemical analysis also demonstrated increased staining of LRG1 in BTC compared with cholangiocytes in benign biliary disease. The combination of receiver operating characteristic (ROC) curves for LRG1, CA19-9 and IL-6 demonstrated an area under the ROC curve of 0.98. In addition, raised LRG1 and CA19-9 were found to be independent predictors of BTC in the presence of elevated bilirubin, C-reactive protein and alkaline phosphatase. CONCLUSION: These results suggest LRG1, CA19-9 and IL-6 as useful markers for the diagnosis of BTC, particularly in high-risk patients with PSC.

Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor.

Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press).

Microarray gene expression and immunohistochemistry analyses of adrenocortical tumors identify IGF2 and Ki-67 as useful in differentiating carcinomas from adenomas.

The management of adrenocortical tumors (ACTs) is complex. The Weiss score is the present most widely used system for ACT diagnosis. An ACT is scored from 0 to 9, with a higher score correlating with increased malignancy. However, ACTs with a score of 3 can be phenotypically benign or malignant. Our objective is to use microarray profiling of a cohort of adrenocortical carcinomas (ACCs) and adrenocortical adenomas (ACAs) to identify discriminatory genes that could be used as an adjunct to the Weiss score. A cohort of Weiss score defined ACCs and ACAs were profiled using Affymetrix HGU133plus2.0 genechips. Genes with high-discriminatory power were identified by univariate and multivariate analyses and confirmed by quantitative real-time reverse transcription PCR and immunohistochemistry (IHC). The expression of IGF2, MAD2L1, and CCNB1 were significantly higher in ACCs compared with ACAs while ABLIM1, NAV3, SEPT4, and RPRM were significantly lower. Several proteins, including IGF2, MAD2L1, CCNB1, and Ki-67 had high-diagnostic accuracy in differentiating ACCs from ACAs. The best results, however, were obtained with a combination of IGF2 and Ki-67, with 96% sensitivity and 100% specificity in diagnosing ACCs. Microarray gene expression profiling accurately differentiates ACCs from ACAs. The combination of IGF2 and Ki-67 IHC is also highly accurate in distinguishing between the two groups and is particularly helpful in ACTs with Weiss score of 3.

Trypanosomiasis: an approach to chemotherapy by the inhibition of carbohydrate catabolism.

When the infected mammalian host of Trypanosoma brucei brucei is injected with a solution of the iron chelator salicyl hydroxamic acid and glycerol, the aerobic and anaerobic glucose catabolism of the parasite is blocked and the parasite is rapidly destroyed.

Role of calcium in trypanocidal drug action.

The synergistic effect of serum on the drug combination of salicylhydroxamic acid plus glycerol, which is active against Trypanosoma brucei, is due to diffusible calcium ions. The synergistic activity can be removed by dialysis of the serum or by addition of calcium chelating agents. A buffer containing calcium can mimic the synergistic activity of serum. This finding may have important implications in the clinical management of African trypanosomiasis in humans. Calcium also has a synergistic effect on melarsoprol, the only drug available for treating sleeping sickness patients with central nervous system involvement, and the concentration of calcium has been reported to be depressed inthe serum of experimentally infected animals.

Rheumatoid factor-like immunoglobulin M protects previously uninfected rat pups and dams from Trypanosoma lewisi.

The serum of lactating rats that have never been infected with the protozoan parasite Trypanosoma lewisi contains a rheumatoid factor-like immunoglobulin M (IgM). This IgM amplifies a specific immunoglobulin G (IgG) response to the parasite and accounts for the unusual resistance of previously uninfected lactating rats and their suckling pups to infection with T. lewisi. A similar rheumatoid factor-like IgM, which is induced late in the usual course of infection with T. lewisi in nonlactating rats, amplifies an earlier IgM response and terminates the infection. To our knowledge, this is the first description of a rheumatoid factor, which is classified as an autoimmune antibody, acting in a protective manner.

Mitochondrial involvement in transhemispheric diaschisis following hypoxia-ischemia: Clomethiazole-mediated amelioration.

Mitochondria play a central role in both the physiological and pathophysiological regulation of cell survival/death. Increasing evidence places mitochondrial dysfunction at the center of many neuropathological conditions. The present study investigates the extent of mitochondrial dysfunction in cortical, hippocampal and cerebellar tissues in a rat model of hypoxia-ischemia (HI). We hypothesized that; mitochondrial dysfunction in situ may be prevented by treatment with clomethiazole (CMZ), a GABA(A) receptor agonist. Assessment of mitochondrial FAD-linked respiration at both 1- and 3-day post-HI revealed a marked decrease in activity from ipsilateral cortical and hippocampal regions (P<0.001). In addition, small changes were seen in contralateral cortical and hippocampal tissues as well as in the cerebellum at 3-days (P<0.05). Assessment of the mitochondrial electron transport chain (complexes I-V), and mitochondrial markers of integrity (citrate synthase) and oxidative stress (aconitase) confirmed mitochondrial impairment in ipsilateral regions following HI. Complexes I, II-III, V and citrate synthase were also impaired in contralateral regions and cerebellum 3-days post-HI. Treatment with CMZ (414 mg/kg/day via minipumps) provided marked protection to all aspects of neuronal tissue assessed. Circulating cytokine (interleukin [IL]-1alpha, IL-1beta, tumor necrosis factor [TNF]-alpha, granulocyte macrophage colony-stimulating factor [GM-CSF], IL-4 and IL-10) levels were also assessed in these animals 3-days post-HI. Plasma IL-1alpha, IL-1beta, TNF-alpha and GM-CSF levels were significantly increased post-HI. Treatment with CMZ ameliorated the increases in IL-1alpha, IL-1beta, TNF-alpha and GM-CSF levels while increasing plasma IL-4 and IL-10 levels. This study provides evidence of the extent of mitochondrial damage following an HI-insult. In addition, we have shown that protection afforded by CMZ extends to preservation of mitochondrial function and integrity via anti-inflammatory mediated pathways.

Dressing remedies: a concept for improving access to and use of dressings in nursing homes.

Delays in accessing dressings prescribed by general practitioners can result in the inappropriate use of dressings on more than one resident in nursing homes. The dressing remedies concept was developed to overcomes this problem.

Dominant situational determinants of sexual risk behaviour in gay men.

OBJECTIVE: To examine the interplay between situational, social and psychological factors in gay men's decisions about HIV risk behaviour, using multiple hypothetical situations. METHOD: The sample included 79 gay men from Sydney recruited through flyers, press advertisements or personal contact at gay venues. Subjects were presented with 20 hypothetical situations graphically on a computer, which included different levels of attraction to partner, intoxication, attractiveness to partner, condom availability, desire for anal sex by subject, insistence of partner on using a condom and time constraints in each situation. Subjects used a computerized graphic rating scale to indicate the likelihood of unprotected anal sex in each situation, the frequency with which they had experienced the hypothetical situation in real life and satisfaction with their response. The probability of having unsafe anal sex in any given situation was the dependent variable. All other variables were predictors. RESULTS: A vector of predictors accounted for 30% of the variance (r = 0.54). Ratings of the probability of anal sex increased with the frequency that subjects had experienced hypothetical situations in real life, attraction to partner, attractiveness to partner and time elapsed since real life experience similar to the hypothetical one. Ratings of the probability of anal sex decreased with condom availability. CONCLUSIONS: Our results suggest that interventions must address the way that individuals interact with their partner and their environment in order to be successful.

A simple and sensitive micro-haemagglutination inhibition assay for the semi-quantitation of the third component of complement.

C3-coated cells have been prepared by incubating sensitised erythrocytes with the appropriate dilutions of serum. Inhibition of agglutination of these cells by an anti-C3 serum provides a sensitive and specific assay for C3 detection and semi-quantitation.

Trypanosoma brucei brucei: patterns of glycolysis at 37 degrees C in vitro.

In the absence of mammalian cells, freshly isolated monomorphic bloodstream forms of Trypanosoma brucei brucei maintain a constant and high level of aerobic glycolysis in vitro for at least 4 h at 37 degrees C when suspended in RPMI medium 1640 containing 20% heat-inactivated and dialyzed fetal calf serum and 25 mM Hepes at an initial pH of 8. In the absence of nutrients other than glucose, salts and protein, some cell death and a decrease in the rate of glycolysis are observed. In the absence of protein, extensive cell death and a decrease in the rate of glycolysis are seen. These observations may be useful in the design of short-term in vitro metabolic studies with T. b. brucei.

p-Alkyloxybenzhydroxamic acids, effective inhibitors of the trypanosome glycerol-3-phosphate oxidase.

Energy production in bloodstream forms of African trypanosomes of the genus Trypanosoma involves two pathways unique to the parasite and which can be blocked by a combination of salicylhydroxamic acid (SHAM) and glycerol. Although this leads to rapid parasite destruction both in vitro and in vivo, the toxicity of SHAM precludes practical use of SHAM/glycerol as a therapeutic regimen. Based on our hypothesis that SHAM operates by interfering with ubiquinone, we attempted to develop this approach by synthesizing and screening a series of hydroxamic acids which more closely resemble ubiquinone: the p-n-alkyloxybenzhydroxamic acids. We also examined a variety of mono-, di- and trisubstituted benzhydroxamic acids together with a selected group of secondary heterocyclic hydroxamic acids. We found an increase in activity of the p-n-alkyloxy compounds with increasing chain length up to 12 carbon atoms with longer chains offering little advantage. The most active compound, p-n-tetradecyloxybenzhydroxamic acid, had an apparent Ki of 0.43 microM indicating a specific activity 70 times greater than SHAM. Although this represents a vast improvement, the low water solubility of these compounds reduces their bioavailability to the point where they are not practical substitutes for SHAM. Consequently, improvement in the SHAM/glycerol approach to chemotherapy appears to lie with improving solubility by altering lipophilicity of the alkyl side chain.

Esters of 3,4-dihydroxybenzoic acid, highly effective inhibitors of the sn-glycerol-3-phosphate oxidase of Trypanosoma brucei brucei.

Alkyl esters of 3,4-dihydroxybenzoic acid are inhibitors of the sn-glycerol-3-phosphate oxidase system of Trypanosoma brucei brucei in vitro and have significant trypanocidal activity in vivo when combined with glycerol. While the parent acid has little inhibitory activity in vitro, the esters are highly active with activity increasing as the chain length of the esterifying alcohol increases. The n-dodecyl ester was more than 400 times as active as salicylhydroxamic acid and 15 times more active than the corresponding p-n-alkyloxybenzhydroxamic acid, one of the most active sn-glycerol-3-phosphate oxidase inhibitors previously reported. When combined with glycerol (to block an alternative pathway of glycolysis) and tested in vitro against intact parasites, this ester was 100 times more effective than salicylhydroxamic acid and 10 times more effective than p-n-dodecyloxybenzhydroxamic acid. It was also active against T. b. brucei in mice when combined with glycerol whereas the latter compound was not. Esters of 3,4,5-trihydroxybenzoic acid (gallic acid) were also highly active while those of 2,3-dihydroxybenzoic acid were much less inhibitory and those of 2,5-dihydroxybenzoic acid were inactive. A related compound, 2',4',5'-trihydroxybutyrophenone, was also active as predicted by its structure but was too toxic to be of interest as a drug candidate.