Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons

Hydrogen sulfide (H₂S) is a gaseous neuromodulator produced from L‐cysteine. H₂S is generated by three distinct enzymatic pathways mediated by cystathionine γ‐lyase (CSE), cystathionine β‐synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H₂S production in PC12 cells (rat pheochromocytoma‐derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H₂S were produced from L‐cysteine in the presence of α‐ketoglutarate, together with dithiothreitol. The production of H₂S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L‐aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H₂S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H₂S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H₂S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H₂S generation.

     

Elastic behavior of a red blood cell with the membrane’s nonuniform natural state: equilibrium shape,motion transition under shear flow,and elongation during tank-treading motion

Direct numerical simulations of the mechanics of a single red blood cell (RBC) were performed by considering the nonuniform natural state of the elastic membrane. A RBC was modeled as an incompressible viscous fluid encapsulated by an elastic membrane. The in-plane shear and area dilatation deformations of the membrane were modeled by Skalak constitutive equation, while out-of-plane bending deformation was formulated by the spring model. The natural state of the membrane with respect to in-plane shear deformation was modeled as a sphere ( \(\alpha =0\) ), biconcave disk shape ( \(\alpha =1\) ) and their intermediate shapes ( \(0<\alpha <1\) ) with the nonuniformity parameter \(\alpha \) , while the natural state with respect to out-of-plane bending deformation was modeled as a flat plane. According to the numerical simulations, at an experimentally measured in-plane shear modulus of \(2.5\times 10^{-6}\,\hbox {N}/\hbox {m}\) and an out-of-plane bending rigidity of \(2.0\times 10^{-19}\,\hbox {N}\cdot \hbox {m}\) of the cell membrane, the following results were obtained. (i) The RBC shape at equilibrium was biconcave discoid for \(\alpha >0.22\) and cupped otherwise; (ii) the experimentally measured fluid shear stress at the transition between tumbling and tank-treading motions under shear flow was reproduced for \(0.05<\alpha <0.34\) ; (iii) the elongation deformation of the RBC during tank-treading motion from the simulation was consistent with that from in vitro experiments, irrespective of the \(\alpha \) value. Based on our RBC modeling, the three phenomena (i), (ii), and (iii) were mechanically consistent for \(0.22<\alpha <0.34\) . The condition \(0.05<\alpha <0.22\) precludes a biconcave discoid shape at equilibrium (i); however, it gives appropriate fluid shear stress at the motion transition under shear flow (ii), suggesting that a combined effect of \(\alpha \) and the natural state with respect to out-of-plane bending deformation is necessary for understanding details of the RBC mechanics at equilibrium. Our numerical results demonstrate that moderate nonuniformity in a membrane’s natural state with respect to in-plane shear deformation plays a key role in RBC mechanics.     

Rapid determination of isoamyl nitrite in pharmaceutical preparations by flow injection analysis with on‐line UV irradiation and luminol chemiluminescence detection

Isoamyl nitrite is used as a therapeutic reagent for cardiac angina and as an antidote for cyanide poisoning, but it is abused because of its euphoric properties. Therefore, a method to determine isoamyl nitrite is required in many fields, including pharmaceutical and forensic studies. In this study, a simple, rapid and sensitive method for the determination of isoamyl nitrite was developed using a flow injection analysis system equipped with a chemiluminescence detector and on‐line photoreactor. This method is based on on‐line ultraviolet irradiation of isoamyl nitrite and subsequent luminol chemiluminescence detection without the addition of an oxidant. A linear standard curve was obtained up to 1.0 μM of isoamyl nitrite with a detection limit (blank + 3SD) of 0.03 μM. The method was successfully applied to determine isoamyl nitrite content in pharmaceutical preparations. Copyright © 2013 John Wiley & Sons, Ltd.     

Spinocerebellar Ataxia Type 31 Is Associated with “Inserted” Penta-Nucleotide Repeats Containing (TGGAA)n

Spinocerebellar ataxia type 31 (SCA31) is an adult-onset autosomal-dominant neurodegenerative disorder showing progressive cerebellar ataxia mainly affecting Purkinje cells. The SCA31 critical region was tracked down to a 900 kb interval in chromosome 16q22.1, where the disease shows a strong founder effect. By performing comprehensive Southern blot analysis and BAC- and fosmid-based sequencing, we isolated two genetic changes segregating with SCA31. One was a single-nucleotide change in an intron of the thymidine kinase 2 gene (TK2). However, this did not appear to affect splicing or expression patterns. The other was an insertion, from 2.5–3.8 kb long, consisting of complex penta-nucleotide repeats including a long (TGGAA)_n stretch. In controls, shorter (1.5–2.0 kb) insertions lacking (TGGAA)_n were found only rarely. The SCA31 repeat insertion''s length inversely correlated with patient age of onset, and an expansion was documented in a single family showing anticipation. The repeat insertion was located in introns of TK2 and BEAN (brain expressed, associated with Nedd4) expressed in the brain and formed RNA foci in the nuclei of patients'' Purkinje cells. An electrophoretic mobility-shift assay showed that essential splicing factors, serine/arginine-rich splicing factors SFRS1 and SFRS9, bind to (UGGAA)_n in vitro. Because (TGGAA)_n is a characteristic sequence of paracentromeric heterochromatin, we speculate that the insertion might have originated from heterochromatin. SCA31 is important because it exemplifies human diseases associated with “inserted” microsatellite repeats that can expand through transmission. Our finding suggests that the ectopic microsatellite repeat, when transcribed, might cause a disease involving the essential splicing factors.     

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic” quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

There is yet to be a reliable prediction of urolithiasis. To facilitate early diagnosis, a simple and rapid high performance liquid chromatography method with electrochemical detection using disposable copper-nanoparticle-plated electrodes (Cuⁿ-SPE) was developed for multiple detection of creatinine and 4 urolithic organic acids. A total of 206 normal and urolithic human and canine urines and urolith samples were collected for direct analysis of creatinine, cystine, uric acid, oxalic acid, and citric acid without sample cleanup and derivatization processes. Urinary organic acids were separated in 11 min and were devoid of ascorbic acid interference. The detection limits (S/N > 3) were at the nanomolar level with linear dynamic ranges spanning 2–3 orders of magnitude. Recoveries in urine ranged from 99.5% for creatinine to 86.5% for citric acid. The analytical variations (RSD) were less than 6.2% in phosphate buffer and 7.7% in urine. Important differences in organic acid levels/profiles between animal species and among normal and urolithic urines/urolith were unveiled and corresponded well (70–90%) with the urolithic risk in a retrospective assessment. The simplicity and reproducibility of this method using disposable Cuⁿ-SPE has made routine urine analysis possible and can be of great clinical and diagnostic potential in the screening of urolithiasis and abnormal states related to excess secretion of organic acids and amino acids in humans and animals.     

Treatment with FTY720 during the induction or effector phase suppresses the development of experimental allergic conjunctivitis in mice

Purpose

To investigate whether experimental allergic conjunctivitis (EC) can be suppressed by treatment with the immunomodulatory drug FTY720, which reduces the recruitment of effector T cells into inflammatory sites.

Methods

BALB/c mice were actively immunized with ragweed (RW) and then injected intraperitoneally with FTY720 on days 0, 2, 4, 6 and 8 (induction phase treatment) followed by challenge on day 10 with RW-containing eye drops. Alternatively, naïve mice that received RW-primed splenocytes were injected intraperitoneally with FTY720 on days 2 and 4 (effector phase treatment) followed by RW challenge on day 4. Twenty-four hours after RW challenge, conjunctivas and spleens were harvested for histology or immunohistochemistry, and flow cytometric analysis or cytokine assays, respectively.

Results

FTY720 treatment during the induction phase suppressed the conjunctival infiltration of T cells as well as eosinophils and macrophages. The splenocytes from induction phase-treated mice contained significantly less CD4⁺ and CD8⁺ T cells and showed significant suppression of Th2 but not Th1 cytokine production. Effector phase treatment with FTY720 suppressed conjunctival eosinophil infiltration.

Conclusions

These data demonstrate that FTY720 treatment during the induction phase decreases the absolute number of CD4⁺ and CD8⁺ T cells in the spleen and suppresses Th2 cytokine production by splenocytes. This leads to the suppression of EC. FTY720 treatment also suppresses EC when delivered during the effector phase. Thus, FTY720 treatment may be suitable for treating severe forms of vernal keratoconjunctivitis.     

Design of stable α‐helices using global sequence optimization

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix‐coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of α‐helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix‐coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable α‐helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 °C is expected to be ～70–75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Initial establishment and regeneration processes of an outlying isolated <Emphasis Type="Italic">Fagus crenata</Emphasis> Blume forest stand in the northernmost boundary of its range in Hokkaido,northern Japan

We investigated initial establishment and regeneration of an outlying isolated Fagus crenata forest stand at the northernmost boundary of its range in Hokkaido, northern Japan. The study site was located in the Sannosuke beech forest (42°46′48″N, 140°23′43″E), a representative outlying beech stand beyond its continuous range. A rectangular 0.75 ha plot was established on a southwest-facing slope and divided into 300 square sub-plots of 25 m². Within each sub-plot, stems over 5 cm in diameter at breast height (DBH) were identified and measured. Furthermore, the location of stems over 10 cm in DBH (canopy stem) was recorded within each sub-plot, and their increment core samples were extracted. Wood from fallen logs was sampled to estimate the species composition of the coarse woody debris. Micro-relief of the plot was investigated by leveling with compasses for a 2.5 m × 2.5 m grid system. In the plot, the population of F. crenata was divided into three sub-populations by their frequency distribution of age. The oldest sub-population, over 121 years old, had been established in small-localized gap in the plot. The sub-population between 81 and 120 years old and the sub-population less than 80 years old were regenerated after a landslide and windthrow in a 1954 typhoon, respectively. Furthermore, dominant species in the plot shifted from Quercus mongolica var. grosseserrata to F. crenata. Consequently, regeneration of F. crenata, i.e., expansion of forest stands, at the northernmost boundary of its range was primarily dependent on episodic natural disturbance, which may be responsible for the reduction of their migration rate in Hokkaido, northern Japan.