A case of juvenile-onset ankylosing spondylitis effectively treated with tumour necrosis factor-alpha inhibitor agents.

A 15-year-old girl had experienced hip pain at 11 years of age. At 15 years of age, the patient complained of persistent generalised pain. Her rheumatoid factor and serum matrix metalloproteinase-3 levels were below standard values; there were no inflammatory responses, and the human leukocyte antigen test was negative for B27 and positive for B52 and B62. The bath ankylosing spondylitis disease activity index (BASDAI) value was 8.0 at the time of induction and 3.1 at 6 months after the introduction of adalimumab (at a dose of 40 mg). The BASDAI value improved with an increase in the dose of adalimumab to 80 mg at 8 months after the initial introduction of adalimumab (at 40 mg), although it remained at 4.8 at 16 months after the dose increase. The BASDAI value was 2.6 at 6 months, 2.7 at 1 year, and 1.8 at 1.5 years after the introduction of infliximab, indicating that the patient had progressed well without any adverse events. Based on this case, juvenile ankylosing spondylitis is a differential diagnosis for low back pain and generalised pain since childhood. Tumour necrosis factor (TNF) inhibitors were promptly introduced in this case, although it took 4 years from the initial presentation. TNF inhibitors were effective in treating juvenile ankylosing spondylitis in the present case without any adverse events. This case is notable because juvenile onset ankylosing spondylitis is one of the reasons for severe lumbago since childhood and because TNF inhibitors were administered promptly after diagnosis.

Engine performance and emissions evaluation of surfactant-free B30 biodiesel-diesel/water emulsion as alternative fuel.

Malaysia is one of the top exporters of palm oil, and although currently facing fierce resistance towards palm oil imports in some parts of the globe, one of the ways to utilize this commodity is by increasing palm biodiesel content in local commercial diesel. However, due to the oxygen-rich nature of biodiesel, its utilization suffers from increased nitrogen oxides (NOx) emission compared to conventional diesel. To mitigate this issue and improve diesel engine performance and emissions using biodiesel-diesel blends, this study attempted to investigate implementation of a real-time non-surfactant emulsion fuel supply system (RTES) which produces water-in-diesel emulsion as fuel without surfactants. NOx reducing capability of water-in-diesel produced by RTES has been well documented. Therefore, in this study, 30% biodiesel-diesel (B30) was used as the base fuel while B30-derived emulsions consisting of 10 wt%, 15 wt% and 20 wt% water content were supplied into a 100 kVA, 5.9-L common rail turbocharged diesel engine electric generator. Fuel consumption and exhaust emissions were measured and compared with commercially available Malaysian low grade diesel fuel (D2M). Evidence suggested that emulsified B30 biodiesel-diesel produced by RTES was able to increase brake thermal efficiency (BTE) up to a maximum of 36% and reduce brake specific fuel consumption (BSFC) up to 8.70%. Furthermore, B30 biodiesel-diesel emulsions produced significantly less NOx, carbon monoxide and smoke at high engine load. In conclusion, B30 biodiesel-diesel emulsions can be readily utilized in current diesel engines without compromising on performance and emissions.

Risk Factors for Incidence of Postoperative Spinal Epidural Hematoma Following Multilevel Microendoscopic Laminectomy.

INTRODUCTION: Due to the narrow portal of entry, microendoscopic laminectomy (MEL) is associated with a risk of postoperative spinal epidural hematoma (POSEH). This risk might be higher when performing multiple-level (m-) MEL. The purpose of this study is to clarify the incidence rate of POSEH following single-level (s-) and m-MEL by each interlaminar level and identify the risk factors for POSEH following m-MEL. METHODS: A total of 379 patients underwent MEL of the lumbar spine (s-MEL, n=141; m-MEL, n=238). We determined the incidence of POSEH following s-MEL and m-MEL by each interlaminar level. For m-MEL, we clarified the correlation between POSEH and possible risk factors, such as operative findings, the sequence of operated interlaminar levels, and the preoperative cross-sectional dural area (CSA) on magnetic resonance imaging. RESULTS: The incidence rate at L2/3 was significantly higher than that at L3/4 and L4/5. Patients who underwent L2/3 decompression at the end of the procedure showed a higher incidence of POSEH at the L2/3 level. Preoperative spinal stenosis was associated with POSEH at the L2/3 level, and CSA of 56 mm2 was a predictive factor for POSEH. Logistic regression analysis revealed that both were significant risk factors. CONCLUSIONS: In patients undergoing m-MEL, the incidence of POSEH is highest at the L2/3 level, and treatment of the L2/3 level at the end of the procedure and the presence of spinal stenosis are risk factors for POSEH.

History of Phos-tag technology for phosphoproteomics.

Phos-tag is a functional molecule that selectively captures a phosphate monoester dianion in neutral aqueous solutions. The affinity of Phos-tag for phosphate monoester dianions is more than 10,000 times greater than that for other anions present in living organisms, such as carboxylic acid anions. We have developed and applied useful techniques for phosphoproteomics based on Phos-tag. This review describes the history of Phos-tag development and outlines three main technologies that have been put to practical use. The first is a technique to separate and concentrate phosphopeptides and phosphoproteins using a Phos-tag derivative with a hydrophilic chromatography carrier (Phos-tag polymer beads). The second is a technology to detect phosphopeptides and phosphoproteins on various arrays using Phos-tag biotin. The third is a technique to separate and detect phosphoproteins by electrophoresis using Phos-tag acrylamide. We hope that these three technologies will make a significant contribution to phosphoproteomics and, ultimately, to life science research. SIGNIFICANCE: The authors found that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acted as a novel phosphate-binding tag nanomolecule, Phos-tag, in an aqueous solution under near physiological conditions. The metal complex having a vacancy on two metal ions is suitable for the access of a phosphomonoester dianion (R-OPO32-) as a bridging ligand. A dinuclear zinc(II) complex (Zn2+-Phos-tag) strongly binds to a p-nitrophenyl phosphate dianion (Kd = 2.5 × 10-8 M) at a neutral pH. The anion selectivity indexes against SO42-, CH3COO-, Cl-, and the bisphenyl phosphate monoanion at 25 °C are 5.2 × 103, 1.6 × 104, 8.0 × 105, and > 2 × 106, respectively. We have been involved in developing technologies by using the Phos-tag molecule and its derivatives to permit the analysis of phosphorylated biomolecules. To date, Phos-tag technology has contributed to the development of several procedures for phosphoproteomics, including a phosphate-affinity chromatography technique for the separation and enrichment of phosphopeptides and phosphoproteins, a wide variety of microarray/on-chip techniques for the detection of protein phosphorylation, and a phosphate-affinity electrophoresis technique for the detection of shifts in the mobilities of phosphoproteins. In this review article, the authors introduce the impact of Phos-tag-based technological advances for phosphoproteomics.

Recent advances in the Phos-tag technique focused on the analysis of phosphoproteins in a bacterial two-component system.

In a bacterial two-component system (TCS), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator (RR). The His- and Asp-bound phosphate groups are extremely unstable under acidic conditions easily to be hydrolyzed within a few hours. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation states in the TCS. Here, we describe that Phos-tag technique is suitable for the quantitative analysis of His- and Asp-phosphorylated proteins. The dynamics of the His-Asp phosphorelay of recombinant TCS derived from Escherichia coli, was examined by Phos-tag SDS-PAGE or Phos-tag fluorescent dye gel staining. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of HK and RR in the presence of ATP or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from HK to RR in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag fluorescent dye gel staining. Consequently, Phos-tag technique provides a simple and convenient approach for screening of HK inhibitors that have potential as new antimicrobial agents. SIGNIFICANCE: Bacterial cells have unique phosphotransfer signaling mechanisms known as two-component systems (TCSs) that permit the organism to sense and respond to various environmental conditions. Each system consists of a histidine kinase (HK) and a response regulator (RR). A typical HK contains an invariant His residue that is autophosphorylated in an ATP-dependent manner. A typical RR has a conserved Asp residue that can acquire a phosphoryl group from its cognate HK. In general, TCS has this type of a His-Asp phosphorelay scheme. Because TCS is also involved in the virulence of pathogens, it is potential targets for novel antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in the bacterial TCS. We believe that our Phos-tag technique provides a simple and convenient approach for drug discovery targeting the bacterial TCS.

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis.

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO2 ) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO2 and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO2 particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO2 particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.

Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems.

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Ascending Aortic Calcification as a Potential Predictor for Low Bone Mineral Density: A Pilot Study.

BACKGROUND: Identifying the factors related to low bone mineral density (BMD) can have significant implications for preventing hip fractures. The correlation between ascending aortic calcification and BMD has never been reported. Therefore, the purpose of the current study is to confirm the hypothesis that ascending aortic calcification can be used as a predictive factor for low BMD and to find a radiographic sign to show it. METHOD: Plain film and computed tomography (CT) images of the thorax were obtained from 91 patients with hip fractures. Using the images, the calcification line of the ascending aorta adjacent to the aortic arch was evaluated. A prominent calcification line confirmed by both plain film and CT was classified as +2. A line which was ambiguous on plain film but confirmed by CT was classified as +1. Cases with no calcification were categorized as 0 (control). We compared the classified score with the BMD and calculated the kappa coefficient to measure intraobserver reliabilities for this radiographic finding. RESULTS: Twenty-eight patients showed a +2 line, twenty-four patients showed a +1 line, and thirty-nine patients showed 0 lines. The median BMD of each group was 0.37 for the +2 line, 0.45 for the +1 line, and 0.51 for the 0 line. The BMD for the +2 group was significantly lower than the others. The kappa coefficient was approximately 0.6 (p < 0.01). CONCLUSION: The imaging finding of calcification of the ascending aorta might be considered as a potential surrogate marker of low BMD. In such subjects, BMD might be ordered for the confirmation of diagnosis of osteoporosis. Mini-Abstract. The Aortic Arch Tail Sign, a calcification line on the ascending aorta, was relevant to low BMD in the current study. BMD can be ordered for the confirmation of diagnosis of osteoporosis in a subject incidentally found to have ascending aorta calcification on X-ray or CT.

An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system.

ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure-activity relationships.

Phos-Tag Fluorescent Gel Staining for the Quantitative Detection of His- and Asp-Phosphorylated Proteins.

We describe a standard protocol for phosphate-affinity fluorescent gel staining that uses a fluorophore-labeled dizinc(II) complex of a derivative of the phosphate-binding tag molecule Phos-tag to detect His- and Asp-phosphorylated proteins separated by SDS-PAGE. The procedure permits the quantitative monitoring of phosphorylated histidine kinases (His-phosphoproteins) and their cognate phosphorylated response regulators (Asp-phosphoproteins) in bacterial two-component signaling transduction systems. The total time required for each gel staining operation is about 2 h at room temperature.

Determining Protein Phosphorylation Status Using Antibody Arrays and Phos-Tag Biotin.

We describe here a standard protocol for determining the phosphorylation status of protein multiplexes using antibody arrays and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer (Phos-tag Biotin). The procedure is based on an antibody microarray technique used in conjunction with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. By using this procedure, we have demonstrated the quantitative detection of the entire phosphorylation status of a target protein involved in intracellular signaling.

Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE.

Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+ Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+ Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and ß-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites. SIGNIFICANCE: Previously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+ Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins. : Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome.

Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic.

Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family (α, γ, δ, and ε) in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic.

Phos-tag-based micropipette-tip method for analysis of phosphomonoester-type impurities in synthetic oligonucleotides.

Various chromatographic techniques, combined with mass spectrometry, have been developed for the analysis of impurities in oligonucleotide drugs, but those methods have generally been less focused on possible phosphomonoester-type compounds. Here, we introduce a simple method for separating terminally phosphorylated impurities from parent oligonucleotides by using a phosphate-affinity micropipette tip (Phos-tag tip). All steps for the phosphate-affinity separation (binding, washing, and elution) are conducted in aqueous buffers at neutral pH. The entire separation protocol requires less than 30 min per sample. In practical examples, we demonstrated that phosphorylated impurities in natural-type and chemically modified oligonucleotides can be efficiently separated by the Phos-tag tip method and subsequently characterized by using ion-pairing reversed-phase liquid chromatography mass spectrometry (IP-RPLC-MS). Thus, a combination of the Phos-tag tip method and IP-RPLC-MS is useful for characterizing and identifying phosphomonoester-type impurities in oligonucleotide drugs.

An immuno-dot blot assay for screening histidine kinase inhibitors.

Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.

A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE.

To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin ß1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes.

In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.

Increase in constitutively active MEK1 species by introduction of MEK1 mutations identified in cancers.

The kinase MEK1 is an essential component of the mitogen-activated protein kinase cascades. Somatic mutations that have been identified in the MEK1-coding gene generally enhance kinase activity. Consequently, MEK1 has attracted much interest as a target for cancer therapy to block the aberrant activity. By using Phos-tag affinity electrophoresis, we found that the introduction of mutations detected in certain sporadic cancers or in MEK-inhibitor-resistant cancer cells produced constitutively active MEK1 species containing phosphorylated Ser-218 and Ser-222 residues; it also enhanced the constitutive activity of the kinase. Phosphorylation profiling of the mutants in the presence of inhibitors of RAF/MEK demonstrated that several mutations conferred resistance to multiple inhibitors as a result of an increase in the quantity of active MEK1 species containing the two phosphorylated Ser-218 and Ser-222 residues. Phos-tag-based phosphorylation profiling of MEK1 can therefore provide clinical insights into characteristics of individual mutations in the MEK1-coding gene.

A method for profiling the phosphorylation state of tyrosine protein kinases.

Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.