Profiling of endogenous metabolites and changes in intestinal microbiota distribution after GEN-001 (Lactococcus lactis) administration.

This study aimed to identify metabolic biomarkers and investigate changes in intestinal microbiota in the feces of healthy participants following administration of Lactococcus lactis GEN-001. GEN-001 is a single-strain L. lactis strain isolated from the gut of a healthy human volunteer. The study was conducted as a parallel, randomized, phase 1, open design trial. Twenty healthy Korean males were divided into five groups according to the GEN-001 dosage and dietary control. Groups A, B, C, and D1 received 1, 3, 6, and 9 GEN-001 capsules (1 × 1011 colony forming units), respectively, without dietary adjustment, whereas group D2 received 9 GEN-001 capsules with dietary adjustment. All groups received a single dose. Fecal samples were collected 2 days before GEN-001 administration to 7 days after for untargeted metabolomics and gut microbial metagenomic analyses; blood samples were collected simultaneously for immunogenicity analysis. Levels of phenylalanine, tyrosine, cholic acid, deoxycholic acid, and tryptophan were significantly increased at 5-6 days after GEN-001 administration when compared with predose levels. Compared with predose, the relative abundance (%) of Parabacteroides and Alistipes significantly decreased, whereas that of Lactobacillus and Lactococcus increased; Lactobacillus and tryptophan levels were negatively correlated. A single administration of GEN-001 shifted the gut microbiota in healthy volunteers to a more balanced state as evidenced by an increased abundance of beneficial bacteria, including Lactobacillus, and higher levels of the metabolites that have immunogenic properties.

Pharmacokinetics and bioequivalence of tofacitinib 5 mg in healthy Korean male subjects.

OBJECTIVE: Tofacitinib is an oral Janus kinase (JAK) inhibitor marketed as an immunomodulator that can effectively treat rheumatoid arthritis. This study aimed to compare the pharmacokinetics and evaluate the bioequivalence of tofacitinib free base (CKD-374) with those of tofacitinib citrate (Xeljanz). MATERIALS AND METHODS: A randomized, open-label, single-dose, 2-sequence, 2-period crossover study was conducted in healthy Korean male subjects. A total of 36 subjects were randomized into two sequence groups. At each period, subjects were administered the test formulation (tofacitinib free base, 5 mg) or the reference formulation (tofacitinib citrate, 8.078 mg; as tofacitinib, 5 mg). The plasma samples were collected up to 12 hours post dose and analyzed by liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters, including maximum plasma concentration (Cmax) and area under the plasma concentration vs. time curve from dosing to the last measurable concentration (AUC0-t), were determined by non-compartmental analysis. The 90% confidence intervals (CIs) of the geometric mean ratios for Cmax and AUC0-t were calculated to evaluate pharmacokinetic equivalence. RESULTS: The 90% CIs of the geometric mean ratios of Cmax and AUC0-t for tofacitinib free base to tofacitinib citrate were 0.9144 - 1.1230 and 1.0245 - 1.0932, respectively. All reported adverse events were of mild intensity, and there were no serious adverse events. CONCLUSION: In healthy Korean male adult subjects, the pharmacokinetic parameters of tofacitinib free base and tofacitinib citrate were evaluated and met the pharmacokinetic bioequivalent criteria. Both formulations were safe and well-tolerated.

Evaluation of the comparative pharmacokinetic properties of a new orally disintegrating tablet of tegoprazan in healthy Korean subjects.

PURPOSE: Tegoprazan is a differentiated gastric acid-pump blocker and belongs to a class of potassium-competitive acid secretion blockers. An orally disintegrating tablet (ODT) of tegoprazan was developed to improve patient compliance. The purpose of this study was to compare pharmacokinetics (PK) and safety profiles between the conventional tablet (as the reference drug) and the ODT (as the test drug) of 50 mg tegoprazan in healthy Korean subjects. MATERIALS AND METHODS: An open-label, randomized, single-dose, 6-sequence, 3-period crossover study was conducted in 48 healthy subjects. All subjects received a single oral dose of tegoprazan 50 mg tablet with water, tegoprazan 50 mg ODT with water, and tegoprazan 50 mg ODT without water. Serial blood samples were collected up to 48 hours after dosing. Plasma concentrations of tegoprazan and its metabolite M1 were measured by LC-MS/MS, and PK parameters were calculated with a non-compartmental method. Safety was evaluated by means of assessed adverse events, physical examinations, laboratory test results as well as measurements of vital signs and ECG throughout the study. RESULTS: A total of 47 subjects completed the study. The 90% confidence intervals of the geometric mean ratios for AUCt, Cmax, and AUCinf of tegoprazan were 0.8873 - 0.9729, 0.8865 - 1.0569, and 0.8835 - 0.9695 for the test drug with water to the reference drug and 0.9169 - 1.0127, 0.9569 - 1.1276, and 0.9166 - 1.0131 for the test drug without water to the reference drug, respectively. There were no serious adverse events, and all adverse events were mild. CONCLUSION: The PK profiles of tegoprazan were equivalent between the conventional tablet and ODT with or without water. There was no significant difference in the safety profiles. Therefore, the novel ODT of tegoprazan that can be taken without water may improve compliance among patients with acid-related diseases.

METORY: Development of a Demand-Driven Blockchain-Based Dynamic Consent Platform Tailored for Clinical Trials.

The recent advent of the dynamic consent concept intensified the data integrity issue in clinical trials. Incorporating blockchain technology into a dynamic consent platform can be a feasible solution. Due to various clinical trial settings, a demand-driven development strategy is required. We developed a blockchain-based dynamic consent platform named METORY tailored for clinical trials. The platform consisted of three parts: web and mobile application user interface, study management platform, and blockchain platform. Hyperledger Fabric, an enterprise-grade private blockchain framework, was used to integrate blockchain into the study consent platform. We conducted user acceptance tests and applied feedback to the improvement of the platform. Identity and role-based access control was constructed by combining mobile-application-based certificate system and access control functionalities in Hyperledger fabric. Data were encrypted using SHA-256 prior to transmission to blockchain server and TLS protocol was used for in-transit encryption. File-system level encryption was separated implemented within the security measures from Amazon RDS. Users' experience in the clinical trial was acceptable in the ease and usefulness of the platform.

Stability of acetylsalicylic acid in human blood collected using volumetric absorptive microsampling (VAMS) under various drying conditions.

Acetylsalicylic acid (ASA) is one of the most commonly used medications in global market, with a risk of intoxication in certain patients. However, monitoring blood drug concentration often requires frequent hospital visits; hence there is an unmet need to increase patient-centricity by conducting blood sampling at home. Volumetric absorptive microsampling (VAMS) is a device that allows collection of homogenous and accurate volume of blood without venipuncture, and can be utilized by patients who are not in hospital settings; but because ASA is prone to hydrolysis and stabilizing reagents cannot be added to VAMS samples, a way to improve sample stability must be developed. The objective of this study was to identify the cause of instability with ASA samples collected by VAMS, and to evaluate ways to improve sample stability. A liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used for analysis of ASA concentration in whole blood. Samples collected with VAMS were kept under different drying conditions (desiccator, pressurized, nitrogen gas and household vacuum sealer) and were compared to the control samples collected by conventional venous sampling. The recovery of ASA was about 31% of the control when VAMS sample was dried at room temperature, whereas VAMS samples under humidity controlled conditions showed more than 85% of recovery. Our results suggest that adequate level of humidity control was critical to ensure sample stability of ASA, and this humidity control could also be achieved at home using household vacuum sealer, thus enabling patient-centric clinical trials to be conducted.

Validation of LC-MS/MS method for determination of rosuvastatin concentration in human blood collected by volumetric absorptive microsampling (VAMS).

In light of the shift toward patient-centric clinical trials, a measure of simplifying blood collection process and minimizing the volume of blood samples is on the rise. Volumetric absorptive microsampling (VAMS) is a microsampling device developed for blood sampling in non-hospital settings, which enables accurate hematocrit-independent collection of 10 or 20 µL of whole blood with a simple finger prick. In this study, liquid chromatography (LC)-tandem mass spectrometry workflow for quantification of rosuvastatin after VAMS sampling was developed and validated. The VAMS sample was stabilized by matrix drying and the optimum LC conditions and extraction methods were used to reach adequate sensitivity with lower limit of quantification verified at 1 ng/mL in 10 µL of blood. The bioanalytical method to quantify rosuvastatin from 1 to 100 ng/mL in VAMS sample was qualified by specificity, carryover, linearity, within-run and between-run reproducibility and stability. Inaccuracy was less than ± 6% and imprecision was less than 10% after analyzing the samples on 5 different days at all concentration levels. In addition, the feasibility of delivery to the analytical laboratory after home sampling during the guaranteed stability period of 10 days at room temperature was confirmed by evaluating concentration changes after VAMS sampling without adding pH buffer. Our results suggest that VAMS sampling did not have an effect on the stability of rosuvastatin, and it is a viable option for simple and accurate blood collection at home.

Pharmacokinetic properties and bioequivalence of gefitinib 250 mg in healthy Korean male subjects.

Gefitinib is an anti-cancer drug used to treat non-small cell lung cancer. The objective of this study was to compare the pharmacokinetics and evaluate the bioequivalence of 2 orally administered gefitinib 250 mg tablets in healthy Korean subjects. A randomized, open-label, single-dose, crossover bioequivalence study was conducted. A total of 50 healthy male volunteers were randomized into 2 sequence groups. During each treatment, the subjects received the test or reference formulation of 250 mg gefitinib with a washout period of 21 days. The plasma samples were collected at pre-dose and up to 144 hours post-dose, and plasma drug concentrations were measured using validated liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were calculated, and the formulations were considered as bioequivalent if the 90% confidence intervals (CIs) of the geometric mean ratios were within the bioequivalence limits of 0.8 to 1.25. Forty-one subjects completed the study and were included in the pharmacokinetic analysis. The 90% CIs of the geometric mean ratios of the test formulation to the reference formulation were 0.8115 to 0.9993 for maximum plasma concentration and 0.9119 to 1.0411 for area under the plasma concentration versus time curve from dosing to the last measurable concentration. There were no serious or unexpected adverse events during the study. In healthy Korean adult subjects, the test and reference formulations of gefitinib 250 mg had similar pharmacokinetic parameters and similar plasma concentration-time profiles. The test formulation of gefitinib met the regulatory criteria for assuming bioequivalence. Both formulations were safe and well-tolerated.

Differential expression of serum proteins in multiple myeloma.

The exact cause instigating multiple myeloma (MM) has not been fully elucidated, and the disease has a median survival of 6 months without any treatment. To identify potential biomarkers of MM, serum proteins reflecting alteration in their proteomes were analyzed in 6 patients with MM compared with 6 healthy controls using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of flight mass spectrometry. The most notable differentially expressed proteins were validated by immunoblotting and changes in mRNA expression were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A total of 11 differentially expressed protein spots were found. The expression levels of 7 proteins [Immunoglobulin heavy constant µ; proto-oncogene diffuse B-cell lymphoma (DBL2); 26S protease regulatory subunit 4 (P26s4); serum albumin; haptoglobin; and two unknown proteins with isoelectronic point (pI) of 6.41 and molecular weight of 35.4 kDa, and pI of 8.05 and molecular weight of 27.4 kDa, respectively] were downregulated in MM compared with healthy controls. Expression of gel actin-related protein 2/3 complex subunit 1A (ARPC1A); immunoglobulin heavy constant γ 1; fibrinogen α chain (FGA) fragment D; and zinc finger protein 70 were increased in serum of MM patients. Protein expressions of ARPC1A, FGA, P26s4 and DBL2 were measured by immunoblotting in an independent cohort of 12 MM patients and 10 healthy controls. RT-qPCR analysis demonstrated that ARPC1A expression only mimicked protein expression, whereas FGA, PSMC1 (encoding P26s4) and MCF2 (encoding DBL2) did not exhibit significant changes in mRNA expression between control and MM samples. These proteins represent putative serological biomarkers for patients with MM.

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

Cyclic ADP-ribose (cADPR) releases Ca2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca2+ ([Ca2+]i) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Comparative proteomic tissue analysis in patients with ossification of the posterior longitudinal ligament.

OBJECTIVE: The ossification of the posterior longitudinal ligament (OPLL) involves the ligament that lines the posterior surface of the spinal vertebral bodies. Hormonal and metabolic factors as well as hereditary factors have been proposed to be involved in pathologic ligamentous OPLL. However, there are currently no definitive serological biomarkers for OPLL that might be used to achieve a more convenient and economic diagnosis. To find an easier and simpler diagnostic method and to identify pathogenic proteins associated with OPLL, we assessed PLL tissues from patients with OPLL for proteomic alterations. METHODS: OPLL tissues were collected from 12 patients with OPLL, and non-OPLL tissues were collected from 12 healthy subjects without OPLL. To minimize individual variations, we matched the sex and age of the patients in the healthy and OPLL groups. The two-dimensional electrophoresis patterns of tissue from 12 OPLL patients and 12 healthy subjects were compared. RESULTS: We found 25 proteins that were significantly and consistently different on the two-dimensional electrophoresis gels between the group of ossified PLL tissues from the patients with OPLL and the group of nonossified PLL tissues from the healthy subjects. Among them, 21 proteins were up-regulated in the patients with OPLL, whereas the remaining four proteins were down-regulated. CONCLUSIONS: The information obtained via this proteomic analysis will be very useful in understanding the pathophysiology of OPLL as well as in finding protein candidates to serve as new diagnostic biomarkers of OPLL.

Proteomic evaluation to identify biomarkers for carpal tunnel syndrome: a comparative serum analysis.

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment, causing pain, impairment, and disability. To identify proteins of CTS comprehensively, a comparative serum analysis of CTS patients and normal control subjects was performed. The two-dimensional electrophoresis patterns of serum obtained from six CTS patients and six normal control subjects were compared. We found 10 proteins that were significantly altered in the serum of CTS patients, among which four were upregulated and six were downregulated. The upregulated spots were identified as Chain A, heat shock 70-kDa protein, 42-kDa ATPase N-terminal domain; glutathione-insulin transhydrogenase (216AA); cAMP-dependent protein kinase inhibitor alpha; and mutant ß-globin. The downregulated spots were identified as vitamin D-binding protein (VDBP), fibrinogen gamma chain, apolipoprotein A-IV (ApoA-IV), clusterin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), and one unidentified protein. The information obtained from this proteomic analysis will be very useful in understanding the pathophysiology of CTS and in finding suitable proteins that can serve as new diagnostic biomarkers of CTS.

Dendroaspis natriuretic peptide regulates the cardiac L-type Ca2+ channel activity by the phosphorylation of α1c proteins.

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca(2+) channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca(2+) current (I(Ca,L)) in a concentration dependent manner with a IC(50) of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 µM). DNP did not affect the voltage dependence of activation and inactivation of I(Ca,L). The α(1c) subunit of cardiac L-type Ca(2+) channel proteins was phosphorylated by the treatment of DNP (1 µM), which was completely blocked by KT5823 (1 µM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 ± 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 µM). These results clearly indicate that DNP inhibits the L-type Ca(2+) channel activity by phosphorylating the Ca(2+) channel protein via PKG activation.

Increased αB-crystallin in hypothalamic paraventricular nucleus of rats with myocardial infarction.

The hypothalamus plays an important role in maintaining a homeostasis of the body against stress response. In particular, the paraventricular nucleus of the hypothalamus is a critical region for disorders related to the autonomic nervous system, such as congestive heart failure and hypertension. αB-crystallin is a family of heat shock proteins that are widely expressed in the brain, including in glial cells, astrocytes, oligodendrocytes, and neurons. Many studies have demonstrated that expression level of αB-crystallin is up-regulated and involved in protecting cells from pathological conditions. In the present study, we examined the expression and potential role of αB-crystallin in the paraventricular nucleus (PVN) regions of rats with myocardial infarction (MI). Our results demonstrate that mRNA encoding αB-crystallin and protein for both native and phosphorylate forms (Ser-59) of αB-crystallin was significantly increased in the PVN during MI.

Comparative analysis of serum proteomes of moyamoya disease and normal controls.

OBJECTIVE: The etiology and pathogenesis of moyamoya disease remain unclear. Furthermore, the definitive diagnostic protein-biomarkers for moyamoya disease are still unknown. The present study analyzed serum proteomes from normal controls and moyamoya patients to identify novel serological biomarkers for diagnosing moyamoya disease. METHODS: We compared the two-dimensional electrophoresis patterns of sera from moyamoya disease patients and normal controls and identified the differentially-expressed spots by matrix-assisted laser desorption/ionization-time-of flight mass spectrometry and electrospray ionization quadruple time-of-flight mass spectrometry. RESULTS: We found and analyzed 22 differently-expressed proteomes. Two proteins were up-regulated. Twenty proteins were down-regulated. Complement C1 inhibitor protein and apolipoprotein C-III showed predominantly changed expressions (complement C1 inhibitor protein averaged a 7.23-fold expression in moyamoya patients as compared to controls, while apolipoprotein C-III averaged a 0.066-fold expression). CONCLUSION: Although our study had a small sample size, our proteomic data provide serologic clue proteins for understanding moyamoya disease.

Proteomic analysis in cyclosporin A-induced overgrowth of human gingival fibroblasts.

Cyclosporin A (CsA) has been used as an immunosuppressive drug to prevent organ transplant rejection and to treat autoimmune diseases. CsA has a proliferative effect on human gingival fibroblasts (HGF) in vitro. However, the molecular mechanisms underlying CsA-induced proliferation in HGF remain to be elucidated. This study was aimed to investigate the CsA responsive proteins in HGF using systematic proteomic approach. Cell viability was determined by MTT assay and reactive oxygen species (ROS) was measured by fluorescent spectrometer. Proteins profiled by two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadrupole time-of-flight mass spectrometry (EIQ-TOF MS). To confirm the expression changes of proteins by proteomics analysis, Western blot was performed using specific antibody. CsA increased the cell viability of HGF in a dose- and time-dependent manner. Significantly, seventeen proteins were overexpressed in the CsA-treated HGF, whereas three proteins were found to be expressed less than the untreated cells. The identified proteins were mainly related with cell proliferation, metabolism, and oxidation. The overexpression of peroxiredoxin 1 (Prx 1) confirmed by Western blotting and reduction of cytosolic reactive oxygen species (ROS) levels in the CsA-treated HGF demonstrated that Prx 1 may play a crucial role in the HGF proliferation induced by CsA. Upregulation of Galectin 3 in CsA-treated HGF indicated that it is related to CsA-induced proliferation. These proteomic analysis data will provide an efficient approach in understanding the mechanisms of HGF proliferation by CsA.

Basic fibroblast growth factor increases intracellular magnesium concentration through the specific signaling pathways.

Basic fibroblast growth factor (bFGF) plays an important role in angiogenesis. However, the underlying mechanisms are not clear. Mg(2+) is the most abundant intracellular divalent cation in the body and plays critical roles in many cell functions. We investigated the effect of bFGF on the intracellular Mg(2+) concentration ([Mg(2+)](i)) in human umbilical vein endothelial cells (HUVECs). bFGF increased [Mg(2+)](i) in a dose-dependent manner, independent of extracellular Mg(2+). This bFGF-induced [Mg(2+)](i) increase was blocked by tyrosine kinase inhibitors (tyrphostin A-23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and a phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) did not affect the bFGF-induced [Mg(2+)](i) increase. These results suggest that bFGF increases the [Mg(2+)](i) from the intracellular Mg(2+) stores through the tyrosine kinase/PI3K/PLCgamma-dependent signaling pathways.

Overexpression of junctate induces cardiac hypertrophy and arrhythmia via altered calcium handling.

Junctate-1 is a newly identified integral endoplasmic/sarcoplasmic reticulum Ca2+ binding protein. However, its functional role in the heart is unknown. In the present study, the consequences of constitutively overexpressed junctate in cardiomyocytes were investigated using transgenic (TG) mice overexpressing junctate-1. TG mice (8 weeks old) showed cardiac remodeling such as marked bi-atrial enlargement with intra-atrial thrombus and biventricular hypertrophy. The TG mice also showed bradycardia with atrial fibrillation, reduced amplitude and elongated decay time of Ca2+ transients, increased L-type Ca2+ current and prolonged action potential durations. Time-course study (2-8 weeks) showed an initially reduced SR function due to down-regulation of SERCA2 and calsequestrin followed by sarcolemmal protein expression and cardiac hypertrophy at later age. These sequential changes could well be correlated with the physiological changes. Adrenergic agonist treatment and subsequent biochemical study showed that junctate-1 TG mice (8 weeks old) were under local PKA signaling that could cause increased L-type Ca2+ current and reduced SR function. Junctate-1 in the heart is closely linked to the homeostasis of E-C coupling proteins and a sustained increase of junctate-1 expression leads to a severe cardiac remodeling and arrhythmias.

The effect of papaverine on ion channels in rat basilar smooth muscle cells.

OBJECTIVES: Papaverine has been used in treating vasospasm following subarachnoid hemorrhage (SAH). However, its action mechanism for cerebral vascular relaxation is not clear. Potassium and calcium channels are closely related to the contraction and relaxation of cerebral smooth muscle. Therefore, to identify the role of potassium and calcium channels in papaverine-induced vascular relaxation, we examined the effect of papaverine on potassium and calcium channels in freshly isolated smooth muscle cells from rat basilar artery. METHOD: The isolation of rat basilar smooth muscle cells was performed by special techniques. The whole cell currents were recorded by whole cell patch clamp technique in freshly isolated smooth muscle cells from rat basilar artery. Papaverine was added to the bath solution. RESULTS: Papaverine of 100 microM into bath solution increased the amplitude of the outward K(+) current which was completely blocked by BKCa blocker, IBX (iberiotoxin) and a calcium chelator, BAPTA (1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) in whole cell mode. Papaverine (100 microM) also inhibited L type Ca(2+) current recorded in isolated smooth muscle cells from rat basilar artery. DISCUSSION: These results strongly suggest that Ca(2+)-activated potassium channels and L type Ca(2+) channels may be involved in papaverine-induced vascular relaxation in rat basilar artery.

Comparative analysis of serum proteomes to discover biomarkers for ossification of the posterior longitudinal ligament.

STUDY DESIGN: Serum proteomes from normal subjects and the patients with ossification of the posterior longitudinal ligament (OPLL) were analyzed by using proteomics. OBJECTIVES: To identify novel serologic biomarkers for diagnosing OPLL. SUMMARY OF BACKGROUND DATA: OPLL can compress the spinal cord, and special planning is required for surgeries that are done from the front of the cervical spine. However, the definitive serologic biomarkers for OPLL are still unclear. METHODS: The 2-dimensional electrophoresis patterns of sera from OPLL patients and normal subjects were compared. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization quadruple time-of-flight mass spectrometry. RESULTS: Nine spots that were differentially expressed in the sera of OPLL patients were found and were identified. PRO2675, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid, an unknown protein, chain B of the crystal structure of deoxy-human hemoglobin beta6, pro-apolipoprotein, ALB protein, retinol binding protein and chain A of human serum albumin mutant R218h complexed with thyroxine (3,3',5,5', tetraiodo-L-thyronine), were up-regulated in the sera of OPLL patients, whereas alpha1-microglobulin/bikunin precursor was down-regulated. CONCLUSIONS: These proteins could be used as diagnostic biomarkers of OPLL.

Synthesis of psoralen derivatives and their blocking effect of hKv1.5 channel.

Previously, we found that a furocoumarin derivative, psoralen (7H-furo[3,2-g][1]benzopyran-7-one), blocked a human Kv1.5 potassium channel (hKv1.5) and has a potential antiarrhythmic effect. In the present study, to develop more potent hKv1.5 blockers or antiarrhythmic drugs, we synthesized ten psoralen derivatives and examined their blocking effects on hKv1.5 stably expressed in Ltk cells. Among the newly synthesized psoralen derivatives, three derivatives (Compounds 5, 9 and 10) showed the open channel-blocking effect. Compound 9 among them was the most potent in blocking hKv1.5. We found that compound 9, one of the psoralen derivatives, inhibited the hKv1.5 current in a concentration-, use- and voltage-dependent manner with an IC50 value of 27.4 +/- 5.1 nM at +60 mV. Compound 9 accelerated the inactivation kinetics of the hKv1.5 channel, slowed the deactivation kinetics of hKv1.5 current resulting in a tail crossover phenomenon. Compound 9 inhibited hKv1.5 current in a use-dependent manner. These results indicate that compound 9, one of psoralen derivatives, acts on hKv1.5 channel as an open channel blocker and is much more potent than psoralen in blocking hKv1.5 channel. If further studies were done, compound 9 might be an ideal antiarrhythmic drug for atrial fibrillation.