LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

High resolution NMR for studying lipid hydrolysis and esterification in cod (Gadus morhua) gonads

High resolution NMR was applied to study biochemical changes of lipids in cod (Gadus morhua) gonads during 7 days storage at 4 degrees C. Changes were observed in the (13)C and (1)H resonances of cholesterol which were due to esterification of fatty acids at the hydroxyl position in roe and milt. Furthermore, the (13)C NMR spectra showed that the lipolytic changes in milt and roe where different. New resonances appeared during storage, due to formation of specific free fatty acids, with the corresponding changes in resonances of the esterified carbonyls and glycerols. The highly unsaturated n-3 fatty acids were hydrolysed from the sn-1 and sn-2 position of both phosphatidylcholine and phosphatidylethanolamine in milt. The lipolytical changes in roe were less prominent compared to the changes in milt, however significant levels of sn-1-lysophospholipids was detected both in roe and milt. The current data demonstrate that high resolution NMR may be a suitable method to non-destructively study hydrolysis and esterification reactions occurring in heterogeneous marine lipids in a one step procedure.     

Homogeneity of recombination rate within a conserved region on BTA3 that contains QTL

The D3S20-D3S34-D3S3 region on BTA3 contains quantitative trait loci (QTL) controlling milk production traits. This region also displays extensive conservation of synteny among several species including cattle, humans, mice and sheep. In this study, we evaluated the adjacent intervals D3S20-D3S34 and D3S34-D3S3 for differences in recombination rate (theta) among bulls in order to assess the suitability of population-based estimates of theta for marker assisted selection and to explore the relationship between variation in theta and chromosome breakpoints associated with mammalian evolution. Using sperm typing, thetaD3S20-D3S34 and theta D3S34-D3S3 were estimated for six triply heterozygous bulls. Recombination frequency ranged from 6.2 to 12.5% and from 9.7 to 19.2% for the D3S20-D3S34 and D3S34-D3S3 intervals, respectively. However, significant variation in theta was not detected between bulls for either interval (D3S20-D3S34 chi(2)5 d.f.=2.59, P < 0.90; D3S34-D3S3 chi(2)5 d.f.=3.72, P < 0.75). The observed differences in theta were most readily attributed to differences in allele-specific amplification efficiencies among bulls. Our results suggest that the positions of QTL in this region can be reliably determined from population data and therefore accurate marker-assisted selection can be performed for desirable alleles without concern for variation in theta. Furthermore, when considered with results of earlier studies, these findings support a correlation between the existence of evolutionary breakpoints or chromosome rearrangements and variation in theta.     

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.     

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression

Introduction

Deletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra).

Methods

The effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA.

Results

rhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA.

Conclusion

Our findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.     

PD98059 enhanced insulin, cytokine, and growth factor activation of xanthine oxidoreductase in epithelial cells involves STAT3 and the glucocorticoid receptor

PD98059 and U0126 are organic compound inhibitors frequently used to block the activity of the MEK-1/2 protein kinase. In the present work, promoter activation analyses of xanthine oxidoreductase (XOR) in epithelial cells uncovered the unexpected opposite effect of these inhibitors on activation of XOR. Activation of an XOR-luciferase fusion gene was studied in stably transfected epithelial cells. The XOR reporter gene was activated by the epidermal growth factors (EGF), prolactin, and dexamethasone and by the acute phase cytokines (APC) IL-1, IL-6, and TNFalpha as previously reported for its native gene, and insulin further stimulated activation induced with acute phase cytokines or growth factors. Activation of the proximal promoter was blocked by inhibitors of the glucocorticoid receptor (GR), p38 MAP kinase, and U0126. Unexpectedly, PD98059 activated the promoter and significantly enhanced expression induced by insulin, APC, or growth factors. Analysis of the XOR upstream DNA and proximal promoter revealed primary roles for the GR and STAT3 in mediating the effects of PD98059 on XOR activation and protein complex formation with the promoter. STAT3 phosphotyrosine-705 was rapidly induced by PD98059, dexamethasone, and insulin. XOR activation by PD98059, dexamethasone, or insulin was superinduced by a constitutively active derivative of STAT3, while a dominant negative derivative of STAT3 blocked the enhancing effect of PD98059 on XOR activation. These data demonstrate a previously unrecognized effect of PD98059 on STAT3 and the GR that could have unanticipated consequences when used to infer the involvement of the MEK-1/2 protein kinase.     

Hydrolysis of triglyceride by the whole cell of Pseudomonas putida 3SK in two-phase batch and continuous reactors systems

Batch and continuous hydrolysis of olive oil in an organic-aqueous two-phase system using the live whole cell of Pseudomonas putida 3SK as a source of a lipase is investigated. The strain was not only fully viable and grown well, but also produced extracellular lipase simultaneously. The degree of hydrolysis, depending on olive oil concentration in the solvents, was maximal at 13.5% (w/v) and decreased with the increase of the substrate concentration. At the optimal condition, a degree of hydrolysis higher than 95% was achieved with 24 h at 30 degrees C when the reaction was carried out in a two-phase batch stirred reactor. For long-term operation a continuous stirred reactor was designed. When the reaction was carried out in a continuous stirred reactor, the degree was hydrolysis reached 86% at a dilution rate of 0.2 h(-1). Satisfactory performance of a two-phase bioreactor was obtained in a long-term continous operation, which lasted for at least 30 days by feeding organic solvent containing olive oil and aqueous media separately. (c) 1994 John Wiley & Sons, Inc.     

Deficiency in the anti‐aging gene Klotho promotes aortic valve fibrosis through AMPKα‐mediated activation of RUNX2

Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti‐aging Klotho gene (KL) promotes high‐fat‐diet‐induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho‐deficient (KL^+/?) mice and WT littermates were fed with a high‐fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPKα activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL^+/? mice than of WT mice, indicating that Klotho deficiency promotes HFD‐induced aortic valve fibrosis (AVF). AMPKα activity (pAMPKα) was decreased, while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL^+/? mice fed with a HFD. Treatment with AICAR markedly attenuated HFD‐induced AVF in KL^+/? mice. AICAR not only abolished the downregulation of pAMPKα but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL^+/? mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho‐deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPKα by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol‐induced upregulation of matrix proteins, including collagen I and osteocalcin. In conclusion, Klotho gene deficiency promotes HFD‐induced fibrosis in aortic valves, likely through the AMPKα–RUNX2 pathway.