Long-distance perturbation on Schiff base-counterion interactions by His30 and the extracellular Na+-binding site in Krokinobacter rhodopsin 2.

Krokinobacter rhodopsin 2 (KR2), a light-driven Na+ pump, is a dual-functional protein, pumping protons in the absence of Na+ when K+ or larger alkali metal ions are present. A specific mutation in helix A near the extracellular Na+ binding site, H30A, eliminates its proton pumping ability. We induced structural changes in H30A by altering the alkali metal ion bound at the extracellular binding site, and observed a strong electrostatic interaction between the Schiff base and counterion and torsion around the Schiff base as revealed by solid-state nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies. The strong interaction when His30 was absent and no ion bound at the extracellular binding site disabled retinal reisomerization, as was shown with flash-photolysis, forming a small amount of only a K-like intermediate. This revealed why H30A lacks the proton pumping function. Long-distance perturbation of the binding site and Schiff base revealed that a non-transported ion binding at the extracellular site is essential for pumping.

Stoichiometry and phosphoisotypes of hippocampal AMPA-type glutamate receptor phosphorylation.

It has been proposed that the AMPAR phosphorylation regulates trafficking and channel activity, thereby playing an important role in synaptic plasticity. However, the actual stoichiometry of phosphorylation, information critical to understand the role of phosphorylation, is not known because of the lack of appropriate techniques for measurement. Here, using Phos-tag SDS-PAGE, we estimated the proportion of phosphorylated AMPAR subunit GluA1. The level of phosphorylated GluA1 at S831 and S845, two major sites implicated in AMPAR regulation, is almost negligible. Less than 1% of GluA1 is phosphorylated at S831 and less than 0.1% at S845. Considering the number of AMPAR at each synapse, the majority of synapses do not contain any phosphorylated AMPAR. Also, we did not see evidence of GluA1 dually phosphorylated at S831 and S845. Neuronal stimulation and learning increased phosphorylation, but the proportion was still low. Our results impel us to reconsider the mechanisms underlying synaptic plasticity.

A possible role of chenodeoxycholic acid and glycine-conjugated bile acids in fibrotic steatohepatitis in a dietary rat model.

BACKGROUND AND AIMS: Our previous study indicated that hepatic bile acids (BAs) may have deposited and stimulated the pathogenesis of a high fat-cholesterol (HFC) diet-induced fibrotic steatohepatitis in stroke-prone spontaneously hypertensive 5/Dmcr rats, based on dysregulated BA homeostasis pathways. We aimed to further characterize BA profiles in liver and evaluate their relationships to liver injury using this model. METHODS: Hepatic 21 BA levels were determined by ultra-performance liquid chromatography-tandem mass spectrometry, and their correlations with macrovesicular steatosis score, serum alanine aminotransferase (ALT) level and quantified fibrotic area were assessed using Spearman and Pearson correlations. RESULTS: Compared to control, BAs highly accumulated in HFC-fed rat liver at 2 weeks: cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) were major species, thereafter, levels of CA and DCA declined, but CDCA species persistently increased, which induced a decrease in total CA/total CDCA ratio at 8 and 14 weeks. CDCA species positively, while total CA/total CDCA negatively, correlated with macrovesicular steatosis score, serum ALT and quantified fibrotic area. Unlike control, total ursodeoxycholic acid was minor in HFC-fed rat liver, and inversely correlated to aforementioned indicators of liver injury; total glyco-BAs, rather than tauro-BAs, were predominant in HFC-fed rat liver, and positively correlated with macrovesicular steatosis score. Moreover, its ratio to total tauro-BAs positively correlated with each parameter of liver injury, while inverse associations were detected for total tauro-BAs. CONCLUSIONS: Hepatic BA accumulation may potentiate liver disease. CDCA and glyco-BAs play a more important role in the pathogenesis of fibrotic steatohepatitis.

Simple and rapid quantitation of 21 bile acids in rat serum and liver by UPLC-MS-MS: effect of high fat diet on glycine conjugates of rat bile acids.

In this report, we present a simple and rapid method for analysis of 21 kinds of bile acids and the conjugates in rat serum and liver samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) in the negative ionization mode, using cholic-2, 2, 4, 4-d4 acid as internal standard. After liquid-liguid extraction from serum and liver samples, specimens were analyzed by UPLC equipped with an Acquity TQD tandem quadrupole mass spectrometer. All of the 21 bile acids were sufficiently separated within 5 min. For most bile acids, calibration curves showed good linearities in the range of 0.25 to 5000 ng/mL for serum samples, 2.5 ng/g to 50 microg/g for liver samples. The limits of detection (LOD) were estimated to be less than 0.25 to 7.5 ng/mL in serum, less than 2.5 to 10 ng/g in liver samples. The present method was validated with respect to repeatability; the coefficient of variation (CV) values were less than 26.7% in the serum and 25.9% in the liver. In the animal study, we compared 21 bile acids in the serum and liver samples of the stroke-prone spontaneously hypertensive (SHRSP) rats fed with control (SP) diet or high-fat and high-cholesterol-containing (HFC) diet. By feeding with HFC diet, the glycine conjugates of some bile acids significantly increased and the taurine conjugate of ulsodeoxicolate (TUDC) decreased in serum and liver samples. Our results suggest that the change of bile acid profiles could be applied for the diagnosis of non-alcoholic fatty liver disease (NAFLD).

Diagnosis of fulminant type 1 diabetes mellitus in an autopsy case with postmortem changes.

A Japanese female in her thirties was found dead in her apartment; the postmortem interval was estimated to be approximately 3 days. Several postmortem changes were evident. Acute gastroenteritis had been diagnosed 3 days earlier. On autopsy, no specific findings other than fatty liver were observed. On hematoxylin eosin staining, mild fibrosis, and invasion of neutrophils in the pancreatic parenchyma, severe fatty liver, and extensive vacuolation of renal tubular cells were observed. Biochemical analyses revealed extremely high ß-hydroxybutyrate concentrations in body fluids with moderate elevation of hemoglobin A1c. Toxicological analyses of organ and urine samples were negative. We concluded that severe ketoacidosis had occurred in the deceased. Subsequently, selective destruction of pancreatic ß-cells was demonstrated. Considered together, results indicated that the cause of death was fulminant type 1 diabetes mellitus. This report illustrates the fact that a combination of biochemical and immunohistochemical investigations can be useful for diagnosing this condition in cases with evident postmortem changes.

High-throughput and simultaneous analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry in the positive and negative ionization modes.

In this report, a high-throughput and sensitive method for analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in the positive and negative ionization modes using tolbutamide as internal standard is presented. After pretreatment of a plasma sample by solid-phase extraction with an Oasis HLB cartridge, muscle relaxants were analyzed by UPLC with Acquity UPLC BEH C(18) column and Acquity TQD tandem quadrupole mass spectrometer equipped with an electrospray ionization interface. The calibration curves for muscle relaxants spiked into human plasma equally showed good linearities in the nanogram per milliliter order range. The detection limits (signal-to-noise ratio = 3) was as low as 0.1-2 ng/mL. The method gave satisfactory recovery rates, accuracy, and precision for quality control samples spiked with muscle relaxants. To further validate the present method, 250 mg of chlorphenesin carbamate was orally administered to a healthy male volunteer, and the concentrations of chlorphenesin carbamate in plasma were measured 0.5, 1, 2, 4, 6, and 8 h after dosing; their concentrations in human plasma were between 0.62 and 2.44 µg/mL. To our knowledge, this is the first report describing simultaneous analysis of over more than two central-acting muscle relaxants by liquid chromatography-tandem mass spectrometry. This has been realized by the capability of our instrument for simultaneous multiple reaction monitoring of the target compounds in both positive and negative ionization modes. Therefore, the present method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.

Sensitive analysis of blonanserin, a novel antipsychotic agent, in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

A rapid and sensitive method for analysis of blonanserin in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry is presented. After pretreatment of a plasma sample by solid-phase extraction, blonanserin was analyzed by the system with a C(18) column. This method gave satisfactory recovery rates, reproducibility, and good linearity of calibration curve in the range of 0.01-10.0 ng/mL for quality control samples spiked with blonanserin. The detection limit was as low as 1 pg/mL. This method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.

Sensitive analysis of aconitine, hypaconitine, mesaconitine and jesaconitine in human body fluids and Aconitum tubers by LC/ESI-TOF-MS.

The Aconitum species (Ranunculaceae) are widely distributed in northern Asia and North America. Their roots are popularly used in herbal medicines in China and Japan. Many cases of accidental, suicidal and homicidal intoxication with this plant have been reported; some of these were fatal because the toxicity of Aconitum is very high. It is thus important to detect and quantify Aconitum alkaloids in body fluids, with high sensitivity. We have developed a simple and sensitive method for measuring four kinds of Aconitum alkaloids (aconitine, hypaconitine, jesaconitine and mesaconitine) by LC/electrospray (ESI)-time-of-flight (TOF)-MS. For all of them, only molecular ions were observed at an orifice voltage of 75 V; at 135 V, base peaks corresponding to [M - 60 + H]+ ions were observed. These four compounds and methyllycaconitine (internal standard) in human plasma samples were purified by solid-phase extraction. The four extracted compounds were completely separated in mass chromatograms; the calibration curves showed good linearity in the range 10-300 ng/ml, and the detection limits were estimated to be 0.2-0.5 ng/ml. Using our method, we also determined the amounts of these compounds in tuber samples. The present method is applicable in clinical and forensic toxicology.

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.