31P NMR spectroscopy demonstrates large amounts of phosphohistidine in mammalian cells

Protein phosphorylation plays a key role in many cellular processes but there is presently no accurate information or reliable procedure to determine the relative abundance of many phosphoamino acids in cells. At pH ≤ 8, phosphohistidine is unstable compared to the extensively studied phosphoserine, phosphothreonine and phosphotyrosine. This study reports the absolute quantitative analysis of histidine phosphorylation of proteins from a human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. The method was designed to minimize loss of the phosphohistidine phosphoryl group. Phosphohistidine was determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 20 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 μmol/g and 68 μmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. After tryptic digest of proteins from the16HBE14o- cell lysate, the phosphohistidine signal was abolished and increasing phosphoserine/phosphothreonine signal was observed, which has implications for mass spectrometry investigations. The 31P NMR spectroscopic analysis not only highlights the abundance of phosphohistidine, which likely reflects its importance in mammalian cells, but also provides a way of measuring and comparing levels of phosphorylated amino acids in cells

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex

Background: The endothelial angiopoietin/Tie2 system is an important regulator of endothelial permeability and targeting Tie2 reduces hemorrhagic shock-induced organ edema in males. However, sexual dimorphism of the endothelium has not been taken into account. This study investigated whether there are sex-related differences in the endothelial angiopoietin/Tie2 system and edema formation.Methods: Adult male and female heterozygous Tie2 knockout mice (Tie2+/−) and wild-type controls (Tie2+/+) were included (n = 9 per group). Renal and pulmonary injury were determined by wet/dry weight ratio and H&amp;E staining of tissue sections. Protein levels were studied in plasma by ELISA and pulmonary and renal mRNA expression levels by RT-qPCR.Results: In Tie2+/+ mice, females had higher circulating angiopoietin-2 (138%, p&lt;0.05) compared to males. Gene expression of angiopoietin-1 (204%, p&lt;0.01), angiopoietin-2 (542%, p&lt;0.001) were higher in females compared to males in kidneys, but not in lungs. Gene expression of Tie2, Tie1 and VE-PTP were similar between males and females in both organs. Renal and pulmonary wet/dry weight ratio did not differ between Tie2+/+ females and males. Tie2+/+ females had lower circulating NGAL (41%, p&lt;0.01) compared to males, whereas renal NGAL and KIM1 gene expression was unaffected. Interestingly, male Tie2+/- mice had 28% higher renal wet/dry weight ratio (p&lt;0.05) compared to Tie2+/+ males, which was not observed in females nor in lungs. Partial deletion of Tie2 did not affect circulating angiopoietin-1 or angiopoietin-2, but soluble Tie2 was 44% and 53% lower in males and females, respectively, compared to Tie2+/+ mice of the same sex. Renal and pulmonary gene expression of angiopoietin-1, angiopoietin-2, estrogen receptors and other endothelial barrier regulators was comparable between Tie2+/- and Tie2+/+ mice in both sexes.Conclusion: Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.</p

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex

Background: The endothelial angiopoietin/Tie2 system is an important regulator of endothelial permeability and targeting Tie2 reduces hemorrhagic shock-induced organ edema in males. However, sexual dimorphism of the endothelium has not been taken into account. This study investigated whether there are sex-related differences in the endothelial angiopoietin/Tie2 system and edema formation.Methods: Adult male and female heterozygous Tie2 knockout mice (Tie2+/−) and wild-type controls (Tie2+/+) were included (n = 9 per group). Renal and pulmonary injury were determined by wet/dry weight ratio and H&amp;E staining of tissue sections. Protein levels were studied in plasma by ELISA and pulmonary and renal mRNA expression levels by RT-qPCR.Results: In Tie2+/+ mice, females had higher circulating angiopoietin-2 (138%, p&lt;0.05) compared to males. Gene expression of angiopoietin-1 (204%, p&lt;0.01), angiopoietin-2 (542%, p&lt;0.001) were higher in females compared to males in kidneys, but not in lungs. Gene expression of Tie2, Tie1 and VE-PTP were similar between males and females in both organs. Renal and pulmonary wet/dry weight ratio did not differ between Tie2+/+ females and males. Tie2+/+ females had lower circulating NGAL (41%, p&lt;0.01) compared to males, whereas renal NGAL and KIM1 gene expression was unaffected. Interestingly, male Tie2+/- mice had 28% higher renal wet/dry weight ratio (p&lt;0.05) compared to Tie2+/+ males, which was not observed in females nor in lungs. Partial deletion of Tie2 did not affect circulating angiopoietin-1 or angiopoietin-2, but soluble Tie2 was 44% and 53% lower in males and females, respectively, compared to Tie2+/+ mice of the same sex. Renal and pulmonary gene expression of angiopoietin-1, angiopoietin-2, estrogen receptors and other endothelial barrier regulators was comparable between Tie2+/- and Tie2+/+ mice in both sexes.Conclusion: Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.</p

A genome-wide association study suggests that a locus within the ataxin 2 binding protein 1 gene is associated with hand osteoarthritis: The Treat-OA consortium

To identify the susceptibility gene in hand osteoarthritis (OA) the authors used a two-stage approach genomewide association study using two discovery samples (the TwinsUK cohort and the Rotterdam discovery subset; a total of 1804 subjects) and four replication samples (the Chingford Study, the Chuvasha Skeletal Aging Study, the Rotterdam replication subset and the Genetics, Arthrosis, and Progression (GARP) Study; a total of 3266 people). Five single-nucleotide polymorphisms (SNPs) had a likelihood of association with hand OA in the discovery stage and one of them (rs716508), was successfully confirmed in the replication stage (meta-analysis p = 1.81×10-5). The C allele conferred a reduced risk of 33% to 41% using a case-control definition. The SNP is located in intron 1 of the A2BP1 gene. This study also found that the same allele of the SNP significantly reduced bone density at both the hip and spine (p<0.01), suggesting the potential mechanism of the gene in hand OA might be via effects on subchondral bone. The authors' findings provide a potential new insight into genetic mechanisms in the development of hand OA

Macrophage ABCA2 deletion modulates intracellular cholesterol deposition, affects macrophage apoptosis, and decreases early atherosclerosis in LDL receptor knockout mice

Biopharmaceutic

Novel Genetic Variants for Cartilage Thickness and Hip Osteoarthritis

Osteoarthritis is one of the most frequent and disabling diseases of the elderly. Only few genetic variants have been identified for osteoarthritis, which is partly due to large phenotype heterogeneity. To reduce heterogeneity, we here examined cartilage thickness, one of the structural components of joint health. We conducted a genome-wide association study of minimal joint space width (mJSW), a proxy for cartilage thickness, in a discovery set of 13,013 participants from five different cohorts and replication in 8,227 individuals from seven independent cohorts. We identified five genome-wide significant (GWS, P≤5·0×10−8) SNPs annotated to four distinct loci. In addition, we found two additional loci that were significantly replicated, but results of combined meta-analysis fell just below the genome wide significance threshold. The four novel associated genetic loci were located in/near TGFA (rs2862851), PIK3R1 (rs10471753), SLBP/FGFR3 (rs2236995), and TREH/DDX6 (rs49654

Identification of new susceptibility loci for osteoarthritis (arcOGEN):a genome-wide association study

To access publisher's full text version of this article. Please click on the hyperlink in Additional Links field.Osteoarthritis is the most common form of arthritis worldwide and is a major cause of pain and disability in elderly people. The health economic burden of osteoarthritis is increasing commensurate with obesity prevalence and longevity. Osteoarthritis has a strong genetic component but the success of previous genetic studies has been restricted due to insufficient sample sizes and phenotype heterogeneity. We undertook a large genome-wide association study (GWAS) in 7410 unrelated and retrospectively and prospectively selected patients with severe osteoarthritis in the arcOGEN study, 80% of whom had undergone total joint replacement, and 11,009 unrelated controls from the UK. We replicated the most promising signals in an independent set of up to 7473 cases and 42,938 controls, from studies in Iceland, Estonia, the Netherlands, and the UK. All patients and controls were of European descent. We identified five genome-wide significant loci (binomial test p≤5·0×10(-8)) for association with osteoarthritis and three loci just below this threshold. The strongest association was on chromosome 3 with rs6976 (odds ratio 1·12 [95% CI 1·08-1·16]; p=7·24×10(-11)), which is in perfect linkage disequilibrium with rs11177. This SNP encodes a missense polymorphism within the nucleostemin-encoding gene GNL3. Levels of nucleostemin were raised in chondrocytes from patients with osteoarthritis in functional studies. Other significant loci were on chromosome 9 close to ASTN2, chromosome 6 between FILIP1 and SENP6, chromosome 12 close to KLHDC5 and PTHLH, and in another region of chromosome 12 close to CHST11. One of the signals close to genome-wide significance was within the FTO gene, which is involved in regulation of bodyweight-a strong risk factor for osteoarthritis. All risk variants were common in frequency and exerted small effects. Our findings provide insight into the genetics of arthritis and identify new pathways that might be amenable to future therapeutic intervention.Arthritis Research UK 1803