Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological ...activation of AMPK rapidly inhibited the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser
and Ser
) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser
and Ser
and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser
and Ser
abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Rα/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.
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Exaggerated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling is key to the pathogenesis of pro-inflammatory disorders, such as rheumatoid arthritis ...and cardiovascular diseases. Mutational activation of JAKs is also responsible for several haematological malignancies, including myeloproliferative neoplasms and acute lymphoblastic leukaemia. Accumulating evidence links adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), an energy sensor and regulator of organismal and cellular metabolism, with the suppression of immune and inflammatory processes. Recent studies have shown that activation of AMPK can limit JAK-STAT-dependent signalling pathways via several mechanisms. These novel findings support AMPK activation as a strategy for management of an array of disorders characterised by hyper-activation of the JAK-STAT pathway. This review discusses the pivotal role of JAK-STAT signalling in a range of disorders and how both established clinically used and novel AMPK activators might be used to treat these conditions.
•Over 3 million pet records analysed showing differences in periodontal disease risk.•The majority of dogs diagnosed with periodontal disease weighed under 15 kg.•Risk factors include age, being ...overweight, and time since last scale and polish.•Highlights dogs most at risk on which diagnostic efforts should be focussed.
Despite periodontal disease (PD) being amongst the most common diagnoses in primary-care practice, the disease is generally underdiagnosed. However, the millions of clinical records generated by pet hospitals each year provide unique opportunities to generate insights about disease risk across large numbers of dogs. The objective of this study was to undertake a retrospective analysis of medical records to ascertain which sizes and breeds of dog are most frequently diagnosed with PD. Although data collection regarding PD was not consistent, it was assumed that the same inconsistencies in recording periodontal abnormalities were present across the range of bodyweight, breed categories and breeds.
Over 3 million medical records across 60 breeds of dogs visiting a chain of veterinary hospitals in the United States collected over a 5-year period were analysed. Statistical analysis of a subset of these records found that extra-small (<6.5 kg) breeds of dog were up to five times more likely to be diagnosed with PD than giant breeds (>25 kg) (P <0.0001). The majority of breeds most frequently diagnosed with PD were in the extra-small, small (6.5−9 kg) and medium-small (9−15 kg) breed size categories. Additional risk factors for PD diagnosis included age, being overweight and time since last scale and polish. Veterinarians should consider targeting client education about dental health, and diagnostic efforts, towards canine patients of the small-breed size categories and those with a higher risk of developing PD (e.g. overweight).
To cite this article: Cochrane SA, Salt LJ, Wantling E, Rogers A, Coutts J, Ballmer‐Weber BK, Fritsche P, Fernández‐Rivas M, Reig I, Knulst A, Le T‐M, Asero R, Beyer K, Golding M, Crevel R, Clare ...Mills EN, Mackie AR. Development of a standardized low‐dose double‐blind placebo‐controlled challenge vehicle for the EuroPrevall Project. Allergy 2012; 67: 107–113.
Background: Double‐blind placebo‐controlled food challenge (DBPCFC) is the gold standard for diagnosing food allergy. Standardized materials and protocols are essential for comparing DBPCFC results for multicentre studies such as EuroPrevall. This required the development and piloting of a standardized vehicle and low‐dose protocol for confirming food allergy and determination of minimum eliciting doses (MEDs).
Methods: A low‐dose DBPCFC protocol was developed, with eight titrated protein doses from 3 μg to 1 g. This was delivered using a simple, microbiologically stable food base incorporating allergenic food ingredients manufactured at three sites and centrally distributed to clinical centres. Allergen blinding was assessed by a professional sensory testing panel using a triangle test. Homogeneity and allergen content were confirmed by ELISA and clinical efficacy was assessed in a pilot study, using celeriac and hazelnut as exemplars.
Results: Celeriac and hazelnut ingredients were sufficiently blinded in the dessert. The dessert meals were successfully piloted with hazelnut in allergy clinics in Spain, the Netherlands and Italy and with celeriac and hazelnut in Zurich. The challenges elicited a range of subjective and objective reactions ranging in severity from mild itching of the oral mucosa to bronchospasm.
Conclusions: A standardized challenge vehicle proven to sufficiently blind processed, powdered hazelnut and celeriac ingredients and that can be reproducibly manufactured has been developed. This pilot study shows that the vehicle is promising for the confirmation of food allergy and determination of MEDs in adults and children with body weight >28.8 kg (approximately 7–11 years old).
Initially the resistance to digestion of two cow’s milk allergens, β-casein, and β-lactoglobulin (β-Lg), was compared using a “high-protease assay” and a “low-protease assay” in a single laboratory. ...The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both β-casein and β-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of β-casein digestion in the low-protease assay were slower, β-Lg being pepsin resistant. During duodenal digestion, β-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.
To avoid metal toxicity, organisms have evolved mechanisms including efflux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion ...facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiae delta zinc resistance conferring (zrc)1delta cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in deltazrc1deltacot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. deltazrc1deltacot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long- or short-term Zn exposure. Expression of TgMTP1 in deltazrc1deltacot1 leads to further lowering of Zn accumulation and an increase in Zn efflux from the cells. Expression of TgMTP1 in a V-type ATPase-deficient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn efflux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green fluorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn efflux in plants.
Worldwide more than 400 plant species are now known that hyperaccumulate various trace metals (Cd, Co, Cu, Mn, Ni, and Zn), metalloids (As) and nonmetals (Se) in their shoots. Of these, almost ...one-quarter are Brassicaceae family members, including numerous Thlaspi species that hyperaccumulate Ni up to 3% of there shoot dry weight. We observed that concentrations of glutathione, Cys, and O-acetyl-L-serine (OAS), in shoot tissue, are strongly correlated with the ability to hyperaccumulate Ni in various Thlaspi hyperaccumulators collected from serpentine soils, including Thlaspi goesingense, T. oxyceras, and T. rosulare, and nonaccumulator relatives, including T. perfoliatum, T. arvense, and Arabidopsis thaliana. Further analysis of the Austrian Ni hyperaccumulator T. goesingense revealed that the high concentrations of OAS, Cys, and GSH observed in this hyperaccumulator coincide with constitutively high activity of both serine acetyltransferase (SAT) and glutathione reductase. SAT catalyzes the acetylation of L-Ser to produce OAS, which acts as both a key positive regulator of sulfur assimilation and forms the carbon skeleton for Cys biosynthesis. These changes in Cys and GSH metabolism also coincide with the ability of T. goesingense to both hyperaccumulate Ni and resist its damaging oxidative effects. Overproduction of T. goesingense SAT in the nonaccumulator Brassicaceae family member Arabidopsis was found to cause accumulation of OAS, Cys, and glutathione, mimicking the biochemical changes observed in the Ni hyperaccumulators. In these transgenic Arabidopsis, glutathione concentrations strongly correlate with increased resistance to both the growth inhibitory and oxidative stress induced effects of Ni. Taken together, such evidence supports our conclusion that elevated GSH concentrations, driven by constitutively elevated SAT activity, are involved in conferring tolerance to Ni-induced oxidative stress in Thlaspi Ni hyperaccumulators.
The gas phase of bread, which makes up more than 70% of the final volume of a loaf, has a major influence on its textural and sensory attributes. Controlling the gas phase volume is a major challenge ...as during proving and early stages of baking gas must be captured within bread dough, only being released at the end of baking. The factors important in determining the gas cell structure are discussed, treating the system as a foam. These include (1) the formation of the initial foam structure during mixing, and (2) stabilization of the foam structure, including those factors governing bubble disproportionation and coalescence. There is particular focus on the role that thin films lining the bubbles may play in stabilizing the foam structure of a risen dough. Despite its potential importance, little is known about the surface properties or composition of the aqueous phase of doughs from which the films are thought to form. We summarize current understanding of the role surface properties may play in determining the aerated structure of dough, and hence the textural characteristics of bread as well as its implications for process engineering aspects of the mixing and proving stages of bread production.
The composition and surface properties of dough liquor isolated by ultracentrifugation have been characterised. Addition of ascorbate had no effect and salts only a limited effect, on the yield, ...protein content and composition of the dough liquor. Fourier transform infrared spectroscopy (FT-IR) revealed the presence of proteins, lipids, starch oligosaccharides together with the non-starch polysaccharide, arabinoxylan. At high dilution the dough liquor air:water interface was dominated by protein, with surface tensions of around 55
mN/m and high surface elasticity. As the concentration was increased, surface tensions dropped to around 40
mN/m for undiluted dough liquor. This was accompanied by the interface becoming less elastic, and indicated that dough liquor lipids were interacting and disrupting the protein films in concentrated dough liquor. Dough liquors from de-fatted flours remained elastic and gave surface tension values of around 50–55
mN/m even at low dilution, indicating that removal of the lipids gave rise to a purely protein stabilised interface. Addition of salt to the dough had the greatest effect on the surface properties, both reducing surface tension and reducing surface elasticity, probably because the charge screening effect of the salt improved the dispersion of lipids in the dough liquor, thus enabling it to disrupt the protein films more effectively. These results indicate that the aqueous phase of bread doughs lining the gas cells would give rise to a mixed protein:lipid interface. Such interfaces are unstable, and would contribute to the instability of the foam structure of risen dough. In addition they show that dough ingredients may modify gas cell stability (and hence may affect crumb structure), by altering the composition and properties of the aqueous phase of doughs.
The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2. ...Five novel genes found in the S. cerevisiae genome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2. The homologs are expressed genes in S. cerevisiae, and their expression is metalloregulated as is true with FRE1 and FRE2. Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor. These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system. Expression of FRE3-FRE6 is elevated in AFT1-1 cells and attenuated in aft1 null cells, showing that iron modulation occurs through the Aft1 transcriptional activator. The fifth homolog FRE7 is specifically copper-metalloregulated. FRE7 is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases. The constitutive expression of FRE7 in MAC1 cells and the lack of expression in mac1-1 cells are consistent with Mac1 being the critical transcriptional activator of FRE7 expression. The 5' promoter sequence of FRE7 contains three copper-responsive promoter elements. Two elements are critical for Mac1-dependent FRE7 expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expression. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are separated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G).
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