Negative modulation of the GABAAρ1 receptor function by l‐cysteine

l ‐Cysteine is an endogenous sulfur‐containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson′s and Alzheimer′s disease. l ‐Cysteine can modulate the activity of ionic channels, including voltage‐gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l ‐cysteine on responses mediated by homomeric GABA_Aρ1 receptors, which are known for mediating tonic γ‐aminobutyric acid (GABA) responses in retinal neurons. GABA_Aρ1 receptors were expressed in Xenopus laevis oocytes and GABA‐evoked chloride currents recorded by two‐electrode voltage‐clamp in the presence or absence of l ‐cysteine. l ‐Cysteine antagonized GABA_Aρ1 receptor‐mediated responses; inhibition was dose‐dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration‐response curves for GABA were shifted to the right in the presence of l ‐cysteine without a substantial change in the maximal response. l ‐Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N‐ethyl maleimide. Our results suggest that redox modulation is not involved during l ‐cysteine actions and that l ‐cysteine might be acting as a competitive antagonist of the GABA_Aρ1 receptors.

     

A single amino acid in the second transmembrane domain of GABA ρ subunits is a determinant of the response kinetics of GABAC receptors

The ρ subunits that constitute the γ‐aminobutyric acid (GABA)_C receptors of retinal neurons form a unique subclass of ligand‐gated chloride channels that give rise to sustained GABA‐evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA ρ1B and ρ2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site‐directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABA_C receptors. When the site contains a proline residue, as in wild‐type ρ1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild‐type ρ2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA ρ receptors, e.g., when the serine residue of the ρ2A receptor was changed to proline, the response of the mutant receptor to imidazole‐4‐acetic acid (I4AA) mimicked that of the ρ1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various ρ receptor subunits. It is noteworthy that the proline residue in native ρ1 subunits and the serine residue of ρ2 subunits are well conserved in all species, a good indication that the presence of multiple GABA ρ subunits serves to generate GABA_C receptors that display the wide range of response kinetics observed on various types of retinal neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 67–76, 1999     

Role of oxygen supply in α, ω‐dodecanedioic acid biosynthesis from n‐dodecane by Candida viswanathii ipe‐1: Effect of stirring speed and aeration

α, ω‐Dodecanedioic acid (DC₁₂) usually serves as a monomer of polyamides or some special nylons. During the biosynthesis, oxygenation cascaded in conversion of hydrophobic n‐dodecane to DC₁₂, while the oxidation of n‐dodecane took place in the intracellular space. Therefore, it was important to investigate the role of oxygen supply on the cell growth and DC₁₂ biosynthesis. It was found that stirring speed and aeration influenced the dissolved oxygen (DO) concentration which in turn affected cell growth as well as DC₁₂ biosynthesis. However, the effect of culture redox potential (Orp) level on DC₁₂ biosynthesis was more significant than that of DO level. For DC₁₂ biosynthesis, the first step was to form the emulsion droplets through the interaction of n‐dodecane and the cell. When the stirring speed was enhanced, slits in the surface layer of the emulsion droplets would be increased. Thus, the substances transportation by water through the slits would be intensified, leading to an enhanced DC₁₂ production. Compared with the batch culture at a lower stirring speed (400 rpm) without culture redox potential (Orp) control, the DC₁₂ concentration was increased by 5 times up to 201.3 g/L with Orp controlled above 0 mV at a higher stirring speed (800 rpm).     

Identification of GABAA Receptor Subunits in Rat Retina: Cloning of the Rat GABAA Receptor ρ2-Subunit cDNA

Abstract: We identified GABA_A receptor subunits in rat retina using PCR. The high degree of conservation among previously described members of ligand-gated anion channels in transmembrane domains was used to design degenerate sense and antisense oligonucleotides. These oligonucleotides were used as primers for PCR, which was applied to the rat retina cDNA. Analysis of clones derived from the PCR amplification identified the GABA_Aα₁, β₁, β₃, and γ₂ subunits and the glycine α₁ subunit. In addition, two clones closely related to the human GABA_Aρ-subunit class were obtained. Molecular cloning revealed one of them as the rat counterpart of the human ρ₂ subunit. Northern blot analysis demonstrated the expression of mRNAs for ρ subunits in retina. These results further support the hypothesis that bicuculline-insensitive GABA channels in rat retina are comprised of ρ subunits.     

Cation–π, amino–π, π–π, and H‐bond interactions stabilize antigen–antibody interfaces

The identification of immunogenic regions on the surface of antigens, which are able to stimulate an immune response, is a major challenge for the design of new vaccines. Computational immunology aims at predicting such regions—in particular B‐cell epitopes—but is far from being reliably applicable on a large scale. To gain understanding into the factors that contribute to the antigen–antibody affinity and specificity, we perform a detailed analysis of the amino acid composition and secondary structure of antigen and antibody surfaces, and of the interactions that stabilize the complexes, in comparison with the composition and interactions observed in other heterodimeric protein interfaces. We make a distinction between linear and conformational B‐cell epitopes, according to whether they consist of successive residues along the polypeptide chain or not. The antigen–antibody interfaces were shown to differ from other protein–protein interfaces by their smaller size, their secondary structure with less helices and more loops, and the interactions that stabilize them: more H‐bond, cation–π, amino–π, and π–π interactions, and less hydrophobic packing; linear and conformational epitopes can clearly be distinguished. Often, chains of successive interactions, called cation/amino–π and π–π chains, are formed. The amino acid composition differs significantly between the interfaces: antigen–antibody interfaces are less aliphatic and more charged, polar and aromatic than other heterodimeric protein interfaces. Moreover, paratopes and epitopes—albeit to a lesser extent—have amino acid compositions that are distinct from general protein surfaces. This specificity holds promise for improving B‐cell epitope prediction. Proteins 2014; 82:1734–1746. © 2014 Wiley Periodicals, Inc.     

SEPARATION OF CHLOROPHYLLS c1c2, AND c3 OF MARINE PHYTOPLANKTON BY REVERSED-PHASE-C18-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY1

We separated chlorophylls c₁ c₂, and c₃ of marine phytoplankton together with other pigments by a modification of the commonly applied reversed-phase-C18-high-performance liquid chromatography (RP-C18-HPLC) method. However, the chlorophyll c-like pigment 2,4, Mg-divinylpheoporphyrin as monomethyl ester, co-eluted with chlorophyll c₁. The method involves optimization of the mobile phase by using a very high ion strength solvent in combination with a high carbon loaded RP-C18 column. Fingerprints of the various taxonomic groups of algae can thus be developed in a single run, including separation of the carotenoids lutein and zeaxanthin.     

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gαi proteins

Crystal structures of Gα_i (and closely related family member Gα_t) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Gα_t exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP‐bound state, unlike Gα_i, which exhibits higher basal exchange and a disordered Switch II region in Gα_iGDP structures. Using purified Gα_i and Gα_t, we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Gα proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GαGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation‐π interaction between tryptophan and arginine residues in the Switch II of Gα_i family proteins that mediates the observed red shift in emission maxima. Furthermore, amino‐terminal myristoylation of Gα_i resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Gα proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.     

Molecular identification of ω-amidase, the enzyme that is functionally coupled with glutamine transaminases, as the putative tumor suppressor Nit2

Our purpose was to identify the sequence of ω-amidase, which hydrolyses the amide group of α-ketoglutaramate, a product formed by glutamine transaminases. In the Bacillus subtilis genome, the gene encoding a glutamine transaminase (mtnV) is flanked by a gene encoding a putative ‘carbon-nitrogen hydrolase’. The closest mammalian homolog of this putative bacterial ω-amidase is ‘nitrilase 2’, whose size and amino acid composition were in good agreement with those reported for purified rat liver ω-amidase. Mouse nitrilase 2 was expressed in Escherichia coli, purified and shown to catalyse the hydrolysis of α-ketoglutaramate and other known substrates of ω-amidase. No such activity was observed with mouse nitrilase 1. We conclude that mammalian nitrilase 2 is ω-amidase.     

Enantioselective σ1 receptor binding and biotransformation of the spirocyclic PET tracer 1′‐benzyl‐3‐(3‐fluoropropyl)‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine]

It was shown that racemic (±)‐ 2 [1′‐benzyl‐3‐(3‐fluoropropyl)‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine], WMS‐1813 ] represents a promising positron emission tomography (PET) tracer for the investigation of centrally located σ₁ receptors. To study the pharmacological activity of the enantiomers of 2 , a preparative HPLC separation of (R)‐2 and (S)‐2 was performed. The absolute configuration of the enantiomers was determined by CD‐spectroscopy together with theoretical calculations of the CD‐spectrum of a model compound. In receptor binding studies with the radioligand [³H]‐(+)‐pentazocine, (S)‐2 was thrice more potent than its (R)‐configured enantiomer (R)‐2 . The metabolic degradation of the more potent (S)‐enantiomer was considerably slower than the metabolism of (R)‐2 . The structures of the main metabolites of both enantiomers were elucidated by determination of the exact mass using an Orbitrap‐LC‐MS system. These experiments showed a stereoselective biotransformation of the enantiomers of 2 . Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Molecular Insight and Mode of Inhibition of α‐Glucosidase and α‐Amylase by Pahangensin A from Alpinia pahangensis Ridl.

The inhibition of carbohydrate‐hydrolyzing enzymes in human digestive organs is crucial in controlling blood sugar levels, which is important in treating type 2 diabetes. In the current study, pahangensin A ( 1 ), a bis‐labdanic diterpene characterized previously in the rhizomes of Alpinia pahangensis Ridl ., was identified as an active dual inhibitor for α‐amylase (IC₅₀=114.80 μm ) and α‐glucosidase (IC₅₀=153.87 μm ). This is the first report on the dual α‐amylase and α‐glucosidase inhibitory activities of a bis‐labdanic diterpene. The Lineweaver‐Burk plots of compound 1 indicate that it is a mixed‐type inhibitor with regard to both enzymes. Based on molecular docking studies, compound 1 docked in a non‐active site of both enzymes. The dual inhibitory activity of compound 1 makes it a suitable natural alternative in the treatment of type 2 diabetes.