Measurement of Proteins in Milk and Dairy Products

The purpose of this study was to develop a short, easy procedure to measure five major proteins in milk and to detect concentrations of added protein to dairy products. Combinations of casein or whey protein with nonfat-dry milk were made with concentration ratios from 0:10 to 10:0. Similar mixtures of defatted goat milk with defatted cow milk were prepared. Samples were hydrolyzed in 6 N HCl at 145°C for 4 h and analyzed for amino acid composition. Multiple regression equations were derived to estimate the relative content of whey protein or casein added to nonfat-dry milk and goat milk added to cow milk employing amino acid profiles of whey protein, casein, nonfat-dry milk, goat milk and cow milk. Correlation coefficient values were all greater than .99. Measuring individual concentrations of milk proteins required separating casein and why proteins by reverse phase high performance liquid chromatography on a C3 column. αs-, β-, and κ-casein were separated after dissociating casein micelles with mercaptoethanol and urea. A 40:60 to 0:100 gradient of .15 M sodium chloride/triethylamine (pH 2.5) and 40% acetonitrile was used. Whey proteins, α-lactalbumin and β-lactoglobulin were separated with a 95:5 to 0:100 gradient of .15 M sodium chloride (pH 2.4) and acetonitrile. Eluted proteins were collected from the column, analyzed for purity by electrophoresis, and hydrolyzed in 6 N HCl at 145°C for 4 h. Purified proteins and mixtures of purified proteins were analyzed for amino acid composition. Estimates of individual protein concentrations in mixtures were made by solving simultaneous equations based on amino acid composition using a tektronix 4052 computer

Survival characteristics of monophasic Salmonella Typhimurium 4,[5],12:i:- strains derived from pig feed ingredients and compound feed

peer-reviewedThe presence of Salmonella in animal feed or feed ingredients at the feed mill or on-farm is a cause for concern, as it can be transmitted to food-producing animals and subsequently to humans. The objective of this study was to determine the survival characteristics of five feed ingredient- and feed-derived monophasic Salmonella Typhimurium 4,[5],12:i:- strains. The first part of the study investigated thermal inactivation using an immersed heating coil apparatus. A Weibull model provided a good fit, with low RMSE values (0.04–0.43) and high R2 values (0.93–0.99) obtained. There was considerable inter-strain variation in heat resistance, with D-values ranging from 397.83 to 689 s at 55 °C, 11.35–260.95 s at 60 °C and 1.12 to 6.81 at 65 °C. Likewise, z-values ranged from 2.95 to 5.44 °C. One strain demonstrated a significantly higher thermal tolerance, even though it had been isolated from a meal feed. However, overall the strains investigated do not appear to be that much more heat resistant than Salmonella previously studied. The second part of this study involved assessing the ability of the five Salmonella strains to survive during storage over a 28-day period in pelleted weaner pig feed treated with 0.3% sodium butyrate. While a mean reduction in the Salmonella count of 0.79 log10 CFU was seen in the treated feed during the storage period, a reduction (albeit only 0.49 log10 CFU) was also observed in the control feed. Although there was no overall effect of treatment, sodium butyrate resulted in reductions in Salmonella counts of 0.75 and 0.22 log10 CFU at days 14 and 24 of feed storage, respectively but at the end of the 28-day storage period counts were 0.25 log10 CFU higher in the treated feed. Therefore, the sodium butyrate used appears unsuitable as an agent for feed treatment perhaps due to the protective coating on the particular feed additive used. Overall, the results of this study enhance knowledge about the behaviour and survival characteristics of monophasic S. Typhimurium 4,[5],12:i:- strains in animal feed and may assist the feed industry and pig producers in implementing effective intervention strategies for their control.Teagasc Walsh Fellowship Programme (Ref: 2011010

Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we, for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization–time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141 mM, as determined by response surface methodology. CHG possessed a 65% increase in water solubility and a 2-fold browning resistance and it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice

ADAM10, as the sheddase of the low affinity IgE receptor (CD23), promotes IgE production and thus is a unique target for attenuating allergic disease. Herein, we describe that B cell levels of ADAM10, specifically, are increased in allergic patients and Th2 prone WT mouse strains (Balb/c and A/J). While T cell help augments ADAM10 expression, Balb WT B cells exhibit increased ADAM10 in the naïve state and even more dramatically increased ADAM10 after anti-CD40/IL4 stimulation compared C57 (Th1 prone) WT B cells. Furthermore, ADAM17 and TNF are reduced in allergic patients and Th2 prone mouse strains (Balb/c and A/J) compared to Th1 prone controls. To further understand this regulation, ADAM17 and TNF were studied in C57Bl/6 and Balb/c mice deficient in ADAM10. C57-ADAM10B-/- were more adept at increasing ADAM17 levels and thus TNF cleavage resulting in excess follicular TNF levels and abnormal secondary lymphoid tissue architecture not noted in Balb-ADAM10B-/-. Moreover, the level of B cell ADAM10 as well as Th context is critical for determining IgE production potential. Using a murine house dust mite airway hypersensitivity model, we describe that high B cell ADAM10 level in a Th2 context (Balb/c WT) is optimal for disease induction including bronchoconstriction, goblet cell metaplasia, mucus, inflammatory cellular infiltration, and IgE production. Balb/c mice deficient in B cell ADAM10 have attenuated lung and airway symptoms compared to Balb WT and are actually most similar to C57 WT (Th1 prone). C57-ADAM10B-/- have even further reduced symptomology. Taken together, it is critical to consider both innate B cell levels of ADAM10 and ADAM17 as well as Th context when determining host susceptibility to allergic disease. High B cell ADAM10 and low ADAM17 levels would help diagnostically in predicting Th2 disease susceptibility; and, we provide support for the use ADAM10 inhibitors in treating Th2 disease

Setting the stage for health: Salutogenesis in midwifery professional knowledge in three European countries

There is a lack of systematic evidence concerning health orientation in maternity practice in the current climate of risk avoidance. The midwifery professional project is orientated toward the preservation of normal physiological processes during the maternity episode. This study investigates accounts of midwives who were working in health-orientated birth settings, to examine if and how they frame a health orientation in professional practice. Twenty-seven narrative interviews were conducted with midwives working in pre-, peri-, and postnatal care in different maternity care settings in Switzerland, Austria, and Germany. In-depth and comparative pattern data analyses were conducted. The distinct practice orientation of the participants was revealed in three main concepts, underpinned by a common framework mirroring the three parameters of the Sense of Coherence (comprehensibility, manageability, and meaningfulness) described in Aaron Antonovsky's salutogenic theory. The midwives’ implicit salutogenic knowledge shaped their reported actions in supporting mothers, fathers, and families to have health-promoting experiences in maternity care. These results suggest that an implicit health orientation in maternity care practice can be prefered through examination of the practice reports of midwives working in settings that have a health-promoting philosophy. Implications for midwifery practice and research are discussed. Consideration is given to the relevance of the results for debates about avoiding overtreatment and for the operationalization of salutogenic theory in health care practice

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Using theories of behaviour to understand transfusion prescribing in three clinical contexts in two countries: Development work for an implementation trial

<p>Abstract</p> <p>Background</p> <p>Blood transfusion is an essential part of healthcare and can improve patient outcomes. However, like most therapies, it is also associated with significant clinical risks. In addition, there is some evidence of overuse. Understanding the potential barriers and enablers to reduced prescribing of blood products will facilitate the selection of intervention components likely to be effective, thereby reducing the number of costly trials evaluating different implementation strategies. Using a theoretical basis to understand behaviours targeted for change will contribute to a 'basic science' relating to determinants of professional behaviour and how these inform the selection of techniques for changing behaviour. However, it is not clear which theories of behaviour are relevant to clinicians' transfusing behaviour. The aim of this study is to use a theoretical domains framework to identify relevant theories, and to use these theories to identify factors that predict the decision to transfuse.</p> <p>Methods</p> <p>The study involves two steps: interview study and questionnaire study. Using a previously identified framework, we will conduct semi-structured interviews with clinicians to elicit their views about which factors are associated with waiting and further monitoring the patient rather than transfusing red blood cells. Interviews will cover the following theoretical domains: knowledge; skills; social/professional role and identity; beliefs about capabilities; beliefs about consequences; motivation and goals; memory, attention, and decision processes; environmental context and resources; social influences; emotion; behavioural regulation; nature of the behaviour. The interviews will take place independently in Canada and the UK and involve two groups of physicians in each country (UK: adult and neonatal intensive care physicians; Canada: intensive care physicians and orthopaedic surgeons). We will: analyse interview transcript content to select relevant theoretical domains; use consensus processes to map these domains on to theories of behaviour; develop questionnaires based on these theories; and mail them to each group of physicians in the two countries. From our previous work, it is likely that the theories will include: theory of planned behaviour, social cognitive theory and the evidence-based strategy, implementation intention. The questionnaire data will measure predictor variables (theoretical constructs) and outcome variables (intention and clinical decision), and will be analysed using multiple regression analysis. We aim to achieve 150 respondents in each of the four groups for each postal survey.</p

Distinct effects on mRNA export factor GANP underlie neurological disease phenotypes and alter gene expression depending on intron content

Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.Peer reviewe