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We provide a standard phosphate-affinity SDS-PAGE (Mn 2+ –Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins (∼150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted migration bands on the improved Mn 2+ –Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of electrophoresis requires about 4 h in this protocol.

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.

Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease that primarily affects the synovial membrane, leading to progressive joint destruction. Among RA-related deformities, forefoot deformities are particularly common, causing severe pain, gait disturbances, and a significant decline in patient quality of life. Typical forefoot deformities observed in patients with RA include hallux valgus (HV), hammer toe deformities, and plantar callosities, all of which require appropriate therapeutic intervention. We aimed to evaluate the clinical outcomes of modified Mitchell’s osteotomy with shortening oblique osteotomy (SOO) for forefoot deformities in patients with RA. Methods Twenty-four patients (31 feet) underwent surgery between 2005 and 2023. The cohort included 22 women (29 feet) and two men (2 feet) with a mean age of 59 ± 12 years and disease duration of 20.3 ± 8.7 years. Clinical outcomes were assessed using the Japanese Society for Surgery of the Foot (JSSF) RA Foot and Ankle Scale and radiographic evaluations. Results At a mean follow-up of 10.1 years, the JSSF scale score improved significantly from 57.9 to 77.1 points; HV angle (HVA) significantly improved from 31.7° to 17.1°; and the 1st to 5th intermetatarsal angles (M1M5A) significantly improved from 29.3° to 20.6°. The 28 joint-Disease activity score with erythrocyte sedimentation rate significantly improved from 2.75 to 2.20. Complications included recurrence of callosities in six feet (16.1%), metatarsal phalangeal joint subluxation in 13 feet (8.4%), appearance of HV deformity in 10 feet (32.3%), and infections in two feet (6.5%). No non-union was observed. Conclusions Modified Mitchell’s osteotomy with SOO significantly reduced pain and improved walking ability in patients with RA. The procedure also achieved a remarkable degree of radiographic correction, particularly a reduction in HVA and M1M5A, contributing to improved forefoot alignment. These findings suggest that the procedure provides clear benefits. Careful attention should be paid to potential postoperative complications such as the appearance of HV deformity and infection.

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.

We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled O-phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λex = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions.

The Fanconi anemia pathway is involved in the signaling of DNA damage. Several Fanconi anemia proteins have been identified, but how the pathway is actually activated was unclear. Now, work on chicken DT40 cells indicates that phosphorylation of FANCI at multiple sites triggers FANCD2 monoubiquitination and DNA-damage repair. In response to DNA damage or replication fork stress, the Fanconi anemia pathway is activated, leading to monoubiquitination of FANCD2 and FANCI and their colocalization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in six conserved and clustered Ser/Thr-Gln motifs of FANCI largely abrogate monoubiquitination and focus formation of both FANCI and FANCD2, resulting in loss of DNA repair function. Conversely, FANCI carrying phosphomimic mutations on the same six residues induces constitutive monoubiquitination and focus formation of FANCI and FANCD2, and protects against cell killing and chromosome breakage by DNA interstrand cross-linking agents. We propose that the multiple phosphorylation of FANCI serves as a molecular switch in activation of the Fanconi anemia pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.

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 (NO x ) 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. NO x 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 NO x , 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.

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 β1 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.

Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected].

In road transport, varying fuel flow rates make it hard to maintain a consistent water ratio in non-surfactant emulsion fuels using the Real-Time Non-Surfactant Emulsion Fuel Supply System (RTES). Thus, it becomes more reasonable to establish an appropriate range of water content tailored to a road condition. Therefore, this study aims to evaluate fuel consumption and exhaust emissions of non-surfactant emulsion fuel in light-duty trucks equipped with RTES, focusing specifically on urban conditions. On-road testing and 300-s idling tests were used as the urban conditions to compare diesel with non-surfactant Water-in-Diesel Emulsion (WiDE) fuel with water percentages from low to high concentrations of water, namely WiDE low%, WiDE med%, and WiDE high%. During idling tests, all emulsion variants reduce fuel consumption. WiDE high% exhibits the most substantial NOx reduction of 9.2%. On-road testing reveals comparable WiDE and diesel fuel consumption, despite the RTES increased electrical load. WiDE high% shows an increment for NOx and CO emissions by 11.71% and 202.19%. In conclusion, a 7.4% to 21.1% water content range was suggested for non-surfactant emulsion fuel in urban road conditions.