MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase

A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-l-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-l-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP.     

Infection imaging using [18F]FDG-labelled white blood cell positron emission tomography–computed tomography

Localizing the sites of infection in the body is possible in nuclear medicine using a variety of radiopharmaceuticals that target different components of the infective and inflammatory cascade. Gamma(γ)-emitting agents such as [67Ga]gallium citrate were among the first tracers used, followed by development of positron-emitting tracers like 2-deoxy-2-[18F]fluoro-D-glucose (¹⁸F-FDG). Though these tracers are quite sensitive, they have limited specificity for infection due to their concentration in sites of non-infective inflammation. White blood cells (WBC) labelled with γ or positron emitters have higher accuracy for differentiating the infective processes from the non-infective conditions that may show positivity with tracers such as ¹⁸F-FDG. We present a pictorial review of potential clinical applications of PET/CT using ¹⁸F-FDG labelled WBC.     

Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline–alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na⁺-Cl⁻ cotransporter (NCC) and Na⁺-K⁺-2Cl⁻ cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.     

Association of methylenetetrahydrofolate reductase (<Emphasis Type="Italic">MTHFR</Emphasis>) gene C677T polymorphism with autism: evidence of genetic susceptibility

Autism (MIM 209850) is a heterogeneous neurodevelopmental disease that manifests within the first 3 years of life. Numerous articles reported that dysfunctional folate-methionine pathway enzymes may play an important role in the pathophysiology of autism. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme of this pathway and MTHFR C677T polymorphism reported as risk factor for autism in several case control studies. However, controversial reports were also published. Hence the present meta-analysis was designed to investigate the relationship of the MTHFR C677T polymorphism with the risk of autism. Electronic databases were searched for case control studies with following search terms - ‘MTHFR’, ‘C677T’, in combination with ‘Autism’. Pooled OR with its corresponding 95 % CI was calculated and used as association measure to investigate the association between MTHFR C677T polymorphism and risk of autism. Total of thirteen studies were found suitable for the inclusion in the present meta-analysis, which comprises 1978 cases and 7257 controls. Meta-analysis using all four genetic models showed significant association between C677T polymorphism and autism (OR_Tvs.C = 1.48; 95 % CI: 1.18–1.86; P = 0.0007; OR_{TT + CT vs. CC} = 1.70, 95 % CI = 0.96–2.9, p = 0.05; OR_{TT vs. CC} = 1.84, 95 % CI = 1.12–3.02, p = 0.02; OR_{CT vs.CC} = 1.60, 95 % CI = 1.2–2.1, p = 0.003; OR_{TT vs.CT+CC} = 1.5, 95 % CI = 1.02–2.2, p = 0.03). In total 13 studies, 9 studies were from Caucasian population and 4 studies were from Asian population. The association between C677T polymorphism and autism was significant in Caucasian (OR_Tvs.C = 1.43; 95 % CI = 1.1–1.87; p = 0.009) and Asian population (OR_Tvs.C = 1.68; 95 % CI = 1.02–2.77; p = 0.04) using allele contrast model. In conclusion, present meta-analysis strongly suggested a significant association of the MTHFR C677T polymorphism with autism.     

Detection and monitoring of methotrexate-associated lung injury using serum markers KL-6 and SP-D in rheumatoid arthritis

The applicability of monitoring concentrations of serum KL-6 and serum surfactant protein-D (SP-D) in the detection of methotrexate-associated lung injury (MTX pneumonitis) in patients with rheumatoid arthritis (RA) was investigated. The concentrations of these markers, sequentially measured in two patients with RA complicated with MTX pneumonitis, were increased in accordance with the severity of MTX pneumonitis. Conversely, the concentrations of these markers were decreased with the improvement of MTX pneumonitis, suggesting that the monitoring of these markers could be applicable not only for detecting the onset of MTX pneumonitis, but also for detecting the therapeutic response of MTX pneumonitis.