Analysis of Crude Protein and Allergen Abundance in Peanuts (Arachis hypogaea cv. Walter) from Three Growing Regions in Australia

The effects of plant growth conditions on concentrations of proteins, including allergens, in peanut (Arachis hypogaea L.) kernels are largely unknown. Peanuts (cv. Walter) were grown at five sites (Taabinga, Redvale, Childers, Bundaberg, and Kairi) covering three commercial growing regions in Queensland, Australia. Differences in temperature, rainfall, and solar radiation during the growing season were evaluated. Kernel yield varied from 2.3 t/ha (Kairi) to 3.9 t/ha (Childers), probably due to differences in solar radiation. Crude protein appeared to vary only between Kairi and Childers, whereas Ara h 1 and 2 concentrations were similar in all locations. 2D-DIGE revealed significant differences in spot volumes for only two minor protein spots from peanuts grown in the five locations. Western blotting using peanut-allergic serum revealed no qualitative differences in recognition of antigens. It was concluded that peanuts grown in different growing regions in Queensland, Australia, had similar protein compositions and therefore were unlikely to show differences in allergenicity.

Differential effects of thyroid hormone manipulation and ß adrenoceptor agonist administration on uncoupling protein mRNA abundance in adipose tissue and thermoregulation in neonatal pigs

We have shown that there is significant disparity in the expression of uncoupling proteins (UCP) 2 and 3 between modern-commercial and ancient-Meishan porcine genotypes, commercial pigs also have higher plasma triiodothyronine (T(3)) in on the first day of life. T(3) and the sympathetic nervous system are both known to regulate UCPs in rodents and humans; their role in regulating these proteins in the pig is unknown. This study examined whether thyroid hormone manipulation or administration of a selective beta3 adrenoceptor agonist (ZD) influenced plasma hormones, colonic temperature and UCP expression in adipose tissue of two breeds of pig. To mimic the differences observed in thyroid hormone status, piglets from Meishan and commercial litters were randomly assigned to control (1 ml/kg water), T(3) (10 mg/kg) (Meishan only), methimazole (a commonly used antithyroid drug) (50 mg/kg) (commercial only) or ZD (10 mg/kg) oral administration for the first 4 days of postnatal life. Adipose tissue UCP2/3 mRNA abundance was measured on day 4 using PCR. T(3) administration raised plasma T(3) concentrations and increased colonic temperature on day 4. UCP3 mRNA abundance was higher in Meishan, than commercial piglets (p = 0.042) and was downregulated following T(3) administration (p = 0.014). Irrespective of genotype, ZD increased UCP2 mRNA abundance (Meishan p = 0.05, commercial p = 0.03). Expression of neither UCP2 nor 3 was related to colonic temperature, regardless of treatment. In conclusion, we have demonstrated a dissociation between thyroid hormones and the sympathetic nervous system in the regulation of UCPs in porcine adipose tissue. We have also suggested that expression of adipose tissue UCP2 and 3 are not related to body temperature in piglets.

Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control.

Association between ferritin, high sensitivity c-reactive protein (hsCRP) and relative abundance of Hepcidin mRNA with the risk of type 2 diabetes in obese subjects

Obesity and Type 2 diabetes mellitus share a strong pro-inflammatory profile. It has been observed that iron is a risk factor in the development of type 2 diabetes. The aim of this study was to evaluate the relationship between iron nutritional status and inflammation with the risk of type 2 diabetes development in obese subjects. We studied 30 obese men with type 2 diabetes (OBDM); 30 obese subjects without diabetes (OB) and 30 healthy subjects (Cn). We isolated peripheral mononuclear cells (PMCs) and challenged them with high Fe concentrations. Total mRNA was isolated and relative abundance of TNF-αIL-6 and hepcidin were determined by qPCR. Iron status, biochemical, inflammatory and oxidative stress parameters were also characterized. OBDM and OB patients showed increased hsCRP levels compared to the Cn group. OBDM subjects showed higher levels of ferritin than the Cn group. TNF-α and IL-6 mRNA relative abundances were increased in OBDM PMCs treated with high/Fe. Hepcidin mRNA was increased with basal and high iron concentration. We found that the highest quartile of ferritin was associated with an increased risk of type 2 diabetes when it was adjusted to BMI and HOMA-IR; this association was independent of the inflammatory status. The highest level of hepcidin gene expression also showed a trend of increased risk of diabetes, however it was not significant. Levels of hsCRP over 2 mg/L showed a significant trend of increasing the risk of diabetes. In conclusion, iron may stimulate the expression of pro-inflammatory genes (TNF-α and IL-6), and both hepcidin and ferritin gene expression levels could be a risk factor for the development of type 2 diabetes. Subjects that have an increased cardiovascular risk also have a major risk to develop type 2 diabetes, which is independent of the BMI and insulin resistance state.

Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes

Skeletal muscle displays enormous plasticity to respond to contractile activity with muscle from strength- (ST) and endurance-trained (ET) athletes representing diverse states of the adaptation continuum. Training adaptation can be viewed as the accumulation of specific proteins. Hence, the altered gene expression that allows for changes in protein concentration is of major importance for any training adaptation. Accordingly, the aim of the present study was to quantify acute subcellular responses in muscle to habitual and unfamiliar exercise. After 24-h diet/exercise control, 13 male subjects (7 ST and 6 ET) performed a random order of either resistance (8 × 5 maximal leg extensions) or endurance exercise (1 h of cycling at 70% peak O2 uptake). Muscle biopsies were taken from vastus lateralis at rest and 3 h after exercise. Gene expression was analyzed using real-time PCR with changes normalized relative to preexercise values. After cycling exercise, peroxisome proliferator-activated receptor-γ coactivator-1α (ET ∼8.5-fold, ST ∼10-fold, P < 0.001), pyruvate dehydrogenase kinase-4 (PDK-4; ET ∼26-fold, ST ∼39-fold), vascular endothelial growth factor (VEGF; ET ∼4.5-fold, ST ∼4-fold), and muscle atrophy F-box protein (MAFbx) (ET ∼2-fold, ST ∼0.4-fold) mRNA increased in both groups, whereas MyoD (∼3-fold), myogenin (∼0.9-fold), and myostatin (∼2-fold) mRNA increased in ET but not in ST (P < 0.05). After resistance exercise PDK-4 (∼7-fold, P < 0.01) and MyoD (∼0.7-fold) increased, whereas MAFbx (∼0.7-fold) and myostatin (∼0.6-fold) decreased in ET but not in ST. We conclude that prior training history can modify the acute gene responses in skeletal muscle to subsequent exercise.

Insights into the role and function of L2, the minor capsid protein of papillomaviruses

Human papillomaviruses (HPV) are responsible for the most common human sexually transmitted viral infections, and high-risk types are responsible for causing cervical and other cancers. The minor capsid protein L2 of HPV plays important roles in virus entry into cells, localisation of viral components to the nucleus, in DNA binding, capsid formation and stability. It also elicits antibodies that are more cross-reactive between HPV types than does the major capsid protein L1, making it an attractive potential target for new-generation, more broadly protective subunit vaccines against HPV infections. However, its low abundance in natural capsids-12-72 molecules per 360 copies of L1-limits its immunogenicity. This review will explore the biological roles of the protein, and prospects for its use in new vaccines. © 2009 Springer-Verlag.

Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis

Quantity and timing of protein ingestion are major factors regulating myofibrillar protein synthesis (MPS). However, the effect of specific ingestion patterns on MPS throughout a 12 h period is unknown. We determined how different distributions of protein feeding during 12 h recovery after resistance exercise affects anabolic responses in skeletal muscle. Twenty-four healthy trained males were assigned to three groups (n = 8/group) and undertook a bout of resistance exercise followed by ingestion of 80 g of whey protein throughout 12 h recovery in one of the following protocols: 8 × 10 g every 1.5 h (PULSE); 4 × 20 g every 3 h (intermediate: INT); or 2 × 40 g every 6 h (BOLUS). Muscle biopsies were obtained at rest and after 1, 4, 6, 7 and 12 h post exercise. Resting and post-exercise MPS (l-[ring-(13)C6] phenylalanine), and muscle mRNA abundance and cell signalling were assessed. All ingestion protocols increased MPS above rest throughout 1-12 h recovery (88-148%, P < 0.02), but INT elicited greater MPS than PULSE and BOLUS (31-48%, P < 0.02). In general signalling showed a BOLUS>INT>PULSE hierarchy in magnitude of phosphorylation. MuRF-1 and SLC38A2 mRNA were differentially expressed with BOLUS. In conclusion, 20 g of whey protein consumed every 3 h was superior to either PULSE or BOLUS feeding patterns for stimulating MPS throughout the day. This study provides novel information on the effect of modulating the distribution of protein intake on anabolic responses in skeletal muscle and has the potential to maximize outcomes of resistance training for attaining peak muscle mass.

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state. J Appl Physiol 112: 1805-1813, 2012. First published March 1, 2012; doi:10.1152/japplphysiol.00170.2012.- We made sex-based comparisons of rates of myofibrillar protein synthesis (MPS) and anabolic signaling after a single bout of high-intensity resistance exercise. Eight men (20 ± 10 yr, BMI = 24.3 ± 2.4) and eight women (22 ± 1.8 yr, BMI = 23.0 ± 1.9) underwent primed constant infusions of L-[ring-13C6]phenylalanine on consecutive days with serial muscle biopsies. Biopsies were taken from the vastus lateralis at rest and 1, 3, 5, 24, 26, and 28 h after exercise. Twenty-five grams of whey protein was ingested immediately and 26 h after exercise. We also measured exercise-induced serum testosterone because it is purported to contribute to increases in myofibrillar protein synthesis (MPS) postexercise and its absence has been hypothesized to attenuate adaptative responses to resistance exercise in women. The exercise-induced area under the testosterone curve was 45-fold greater in men than women in the early (1 h) recovery period following exercise (P < 0.001). MPS was elevated similarly in men and women (2.3- and 2.7-fold, respectively) 1-5 h postexercise and after protein ingestion following 24 h recovery. Phosphorylation of mTORSer2448 was elevated to a greater extent in men than women acutely after exercise (P = 0.003), whereas increased phosphorylation of p70S6K1Thr389 was not different between sexes. Androgen receptor content was greater in men (main effect for sex, P = 0.049). Atrogin-1 mRNA abundance was decreased after 5 h recovery in both men and women (P < 0.001), and MuRF-1 expression was elevated in men after protein ingestion following 24 h recovery (P = 0.003). These results demonstrate minor sex-based differences in signaling responses and no difference in the MPS response to resistance exercise in the fed state. Interestingly, our data demonstrate that exerciseinduced increases in MPS are dissociated from postexercise testosteronemia and that stimulation of MPS occurs effectively with low systemic testosterone concentrations in women.

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L-P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N-7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of gamma-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5' sequence. The L protein forms a covalent enzyme-guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717-2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

Structural proteins of mycobacteriophage I3: cloning, expression and sequence analysis of a gene encoding a 70-kDa structural protein

The structural proteins of mycobacteriophage I3 have been analysed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), radioiodination and immunoblotting. Based on their abundance the 34- and 70-kDa bands appeared to represent the major structural proteins. Successful cloning and expression of the 70-kDa protein-encoding gene of phage I3 in Escherichia coli and its complete nucleotide sequence determination have been accomplished, A second (partial) open reading frame following the stop codon for the 70-kDa protein was also identified within the cloned fragment. The deduced amino-acid sequence of the 70-kDa protein and the codon usage patterns indicated the preponderance of codons, as predicted from the high G+C content of the genomic DNA of phage I3.

Typical and atypical domain combinations in human protein kinases: functions, disease causing mutations and conservation in other primates

Ser/Thr and Tyr protein kinases orchestrate many signalling pathways and hence loss in this balance leads to many disease phenotypes. Due to their high abundance, diversity and importance, efforts have been made in the past to classify kinases and annotate their functions at both gross and fine levels. These kinases are conventionally classified into subfamilies based on the sequences of catalytic domains. Usually the domain architecture of a full-length kinase is consistent with the subfamily classification made based on the sequence of kinase domain. Important contributions of modular domains to the overall function of the kinase are well known. Recently occurrence of two kinds of outlier kinases-''Hybrid'' and ``Rogue'' has been reported. These show considerable deviations in their domain architectures from the typical domain architecture known for the classical kinase subfamilies. This article provides an overview of the different subfamilies of human kinases and the role of non-kinase domains in functions and diseases. Importantly this article provides analysis of hybrid and rogue kinases encoded in the human genome and highlights their conservation in closely related primate species. These kinases are examples of elegant rewiring to bring about subtle functional differences compared to canonical variants.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

An assessment of the ability of the stay-green phenotype in Lolium species to provide an improved protein supply for ruminants

Kingston-Smith, A. H., Bollard, A. L., Humphreys, M. O,, Theodorou, M. K. (2002). An assessment of the ability of the stay-green phenotype in Lolium species to provide an improved protein supply for ruminants. Annals of Botany, 89(6), 731-740. Sponsorship: BBSRC/MAFF/Milk Development Council/Meat and Livestock Commission/Industry. RAE2008

Age- and activity-related differences in the abundance of Myosin essential and regulatory light chains in human muscle

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM).We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping.

Unveiling Protein Kinase A Targets in Cryptococcus neoformans Capsule Formation.

The protein kinase A (PKA) signal transduction pathway has been associated with pathogenesis in many fungal species. Geddes and colleagues [mBio 7(1):e01862-15, 2016, doi:10.1128/mBio.01862-15] used quantitative proteomics approaches to define proteins with altered abundance during protein kinase A (PKA) activation and repression in the opportunistic human fungal pathogen Cryptococcus neoformans. They observed an association between microbial PKA signaling and ubiquitin-proteasome regulation of protein homeostasis. Additionally, they correlated these processes with expression of polysaccharide capsule on the fungal cell surface, the main virulence-associated phenotype in this organism. Not only are their findings important for microbial pathogenesis, but they also support similar associations between human PKA signaling and ubiquitinated protein accumulation in neurodegenerative diseases.