Dimeric and trimeric proanthocyanidins possessing a doubly linked structure from Pavetta owariensis

Pavetannin A-2, a new A-type proanthocyanidin, along with the trimers cinnamtannin B-1, pavetannin B-1, B-2, B-3, B-5 and B-6 have been isolated in their free phenolic form from the stem bark of Pavetta owariensis. Spectral data and partial acid-catalysed degradation established their structures as ent-epicatechin-(4→8,2→O→7)-catechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4→8)-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-ent-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-epicatechin, epicatechin-(4β→6,2β→O→7)-epicatechin-(4β→8)-epicatechin, epicatechin-(4β→6,2β→O→7)-catechin-(4β→8)-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4→8)-catechin, respectively.     

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol

Two isoforms of 11β-HSD exist; 11β-HSD1 is bi-directional (the reductase usually being predominant) and 11β-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, “glycyrrhetinic acid-like factors (GALFs)”, which like licorice, inhibit the bi-directional 11β-HSD1 enzyme as well as the dehydrogenase reaction of 11β-HSD2.

Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 35-tetrahydro-corticosterone, its derivative, 35-tetrahydro-11β-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 35-tetrahydro-11β-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11β-HSD1 and 11β-HSD2-dehydrogenase, with IC₅₀'s in range 0.26–3.0 μM, whereas their 11-keto-35-tetrahydro-derivatives inhibit 11β-HSD1 reductase, with IC₅₀'s in range 0.7–0.8 μM (their 35β-derivatives being completely inactive).

Inhibitors of 11β-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11β-HSD1 dehydrogenase/11β-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C₂₁- and C₁₉-steroidal substances may serve as 11β-HSD1- or 11β-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.     

The F1-ATPase beta-subunit is the putative enterostatin receptor

It has been suggested that the F₁-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin_1–7, in the absence and presence of cold enterostatin. ¹²⁵I-β-casomorphin_1–7 weakly binds to the rat F₁-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin_1–7 binding to the F₁-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin_1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin_1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F₁-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F₁-ATPase complex. Western blot analysis showed the F₁-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F₁-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin_1–7 bind to distinct sites on the protein.     

Beta-ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Beta(3)-adrenoceptor agonists for the treatment of frequent urination and urinary incontinence: 2-[4-(2-[[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl)phenoxy]-2-methylpropionic acid.

In a search for novel analogues of β₃-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dysfunction, 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acids (1a–e), into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective β₃-AR agonist in functional assays using the ferret detrusor (β₃-AR), rat uterus (β₂-AR), and rat atrium (β₁-AR); β₃: EC₅₀=7.8 nM, β₂: IC₅₀=7,300 nM, β₁: EC₂₀=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the phenyl ring of 1a further improved β₃-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED₅₀=31 μg/kg) in rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b–e), possess β₃-AR agonistic activity in the absence of undesirable β₁- or β₂-AR mediated actions, and may be useful for clinical treatment and pharmacological studies.     

Enzymatic transglycosylation of xylose using a glycosynthase

The application of the hyperactive glycosynthase derived from Agrobacterium sp. β-glucosidase (AbgE358G-2F6) to the synthesis of xylo-oligosaccharides by using -d-xylopyranosyl fluoride as donor represents the first successful use of glycosynthase technology for xylosyl transfer. Transfer to p-nitrophenyl β-d-glucopyranoside yields di- and trisaccharide products with β-(1→4) linkages in 63% and 35% yields, respectively. By contrast, transfer to p-nitrophenyl β-d-xylopyranoside yielded the β-(1→3) linked disaccharide and β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-β-d-Xyl-pNP as major products in 42% and 30% yields, respectively. Transfer of xylose to β-d-Xyl-(1→4)-β-d-Xyl-pNP yielded the β-(1→4) linked trisaccharide in 98% yield, thereby indicating that transfers to xylo-disaccharides occur with formation of β-(1→4) bonds. Xylosylation of carbamate-protected deoxyxylonojirimycin produced a mixture of di- and tri-‘saccharide’ products in modest yields.     

Phenylethanolamine N-methyltransferase has beta-carboline 2N-methyltransferase activity: hypothetical relevance to Parkinson's disease

Mammalian brain has a β-carboline 2N-methyltransferase activity that converts β-carbolines, such as norharman and harman, into 2N-methylated β-carbolinium cations, which are structural and functional analogs of the Parkinsonian-inducing toxin 1-methyl-4-phenylpyridinium cation (MPP⁺). The identity and physiological function of this β-carboline 2N-methylation activity was previously unknown. We report pharmacological and biochemical evidence that phenylethanolamine N-methyltransferase (EC 2.1.1.28) has β-carboline 2N-methyltransferase activity. Specifically, purified phenylethanolamine N-methyltransferase (PNMT) catalyzes the 2N-methylation (21.1 pmol/h per unit PNMT) of 9-methylnorharman, but not the 9N-methylation of 2-methylnorharmanium cation. LY134046, a selective inhibitor of phenylethanolamine N-methyltransferase, inhibits (IC₅₀ 1.9 μM) the 2N-methylation of 9-methylnorharman, a substrate for β-carboline 2N-methyltransferase. Substrates of phenylethanolamine N-methyltransferase also inhibit β-carboline 2N-methyltransferase activity in a concentration-dependent manner. β-Carboline 2N-methyltransferase activity (43.7 pmol/h/mg protein) is present in human adrenal medulla, a tissue with high phenylethanolamine N-methyltransferase activity.

We are investigating the potential role of N-methylated β-carbolinium cations in the pathogenesis of idiopathic Parkinson’s disease. Presuming that phenylethanolamine N-methyltransferase activity forms toxic 2N-methylated β-carbolinium cations, we propose a novel hypothesis regarding Parkinson’s disease—a hypothesis that includes a role for phenylethanolamine N-methyltransferase-catalyzed formation of MPP⁺-like 2N-methylated β-carbolinium cations.     

A cDNA cloned from Physarum polycephalum encodes new type of family 3 beta-glucosidase that is a fusion protein containing a calx-beta motif

The microplasmodia of Physarum polycephalum express three types of β-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane β-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane β-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane β-glucosidase 1 sequence and were highly homologous with the primary structures of fungal β-glucosidases. Notably, the C-terminal half of membrane β-glucosidase 1 contains two calx-β motifs, which are known to be Ca²⁺ binding domains in the Drosophila Na⁺/Ca²⁺ exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane β-glucosidase 1 differs from all previously identified family 3 β-glucosidases. In addition to cDNA for membrane β-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane β-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other β-glucosidase forms occurred in Physarum poycephalum.

Thus, the membrane β-glucosidase is a new type family 3 enzyme fused with the Calx-β domain. We propose that Calx-β domain may modulate the β-glucosidase activity in response to changes in the Ca²⁺ concentration.     

Chaperone activities of bovine and camel beta-caseins: Importance of their surface hydrophobicity in protection against alcohol dehydrogenase aggregation

β-Casein (β-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel β-CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k_c, μM⁻¹), thermal aggregation constant (k_T, °C⁻¹) and aggregation rate constant (k_t, min⁻¹) were measured. Bovine β-CN displays greater chaperone activity than camel β-CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine β-CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel β-CN. Greater relative hydrophobicity of bovine β-CN than camel β-CN may be a factor responsible for stronger interactions of bovine β-CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine β-CN.     

4-Aminopiperidine ureas as potent selective agonists of the human beta(3)-adrenergic receptor.

The preparation and structure–activity relationships (SARs) of potent agonists of the human β₃-adrenergic receptor (AR) derived from a 4-aminopiperidine scaffold are described. Examples combine human β₃-AR potency with selectivity over human β₁-AR and/or human β₂-AR agonism. Compound 29s was identified as a potent (EC₅₀=1 nM) and selective (greater than 400-fold over β₁- with no β₂-AR agonism) full β₃-AR agonist with in vivo activity in a transgenic mouse model of thermogenesis.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

Probing the opioid receptor complex with (+)-trans-SUPERFIT. II. Evidence that mu ligands are noncompetitive inhibitors of the delta cx opioid peptide binding site.

Previous studies delineated two classes of δ binding sites; a δ binding site not associated with the opioid receptor complex, termed the δ_ncx site, and a δ site associated with the opioid receptor complex, termed the δ_cx site. The δ_ncx site has high affinity for [ -Pen², -Pen⁵]enkephalin, and is synonymous with what is now identified as the δ₁ binding site. Pretreatment of membranes with the δ-selective acylating agents FIT, or (+)-trans-SUPERFIT, deplete membranes of the δ_ncx binding site, which permits the selective labeling of the δ_cx binding site with [³H][ -Ala²,Leu⁵]enkephalin. The present study compared the properties of the δ_cx binding site present in brain membranes pretreated with (+)-trans-SUPERFIT with the properties of the δ_cx site present in untreated membranes. The major findings are: 1) pretreatment of membranes with (+)-trans-SUPERFIT decreased the IC₅₀ values of δ-preferring drugs, and increased the IC₅₀ values of μ-preferring drugs, for the δ_cx binding site; 2) the degree of δ selectivity was highly correlated with the magnitude of the (+)-trans-SUPERFIT-induced shift in the IC₅₀ values; 3) the ligand-selectivity patterns of the μ and δ_cx sites present in (+)-trans-SUPERFIT-pretreated membranes were poorly correlated; 4) whereas μ-preferring drugs were noncompetitive inhibitors of [³H][ -Ala²,Leu⁵]enkephalin binding to the δ_cx site, δ-preferring drugs were competitive inhibitors. Viewed collectively, these data support the hypothesis that the μ and δ_cx binding sites are distinct, provide additional evidence for δ receptor heterogeneity, and suggest that ( (+)-trans-SUPERFIT-pretreated membranes will provide a useful preparation for studying the δ_cx binding site.     

Some observations on the measurement of estradiol-17β in human plasma by radioimmunoassay

The use of specific and non-specific antisera for estradiol-17β (E₂17β) were compared in the radioimmunoassay of the steroid. The effects of various “blank” mateirials on the standard curve and on the accuracy of recovery of E₂17β added to plasma before and after chromatography on LH-20 Sephadex were examined. It was concluded that the use of the specific antiserum (anti-6-oxoE₂17β -6-(O-carboxymethyl)oxime-bovine serum albumin(antiE₂17β-6-BSA) was an improvement on the non-specific serum anti-E₂17β-17-hemisuccinyl-bovine serum albumin (antiE₂ 17β-17-BSA) following chromatography of extracts. However, although a precise result could be obtained with the anti-E₂17β-6-BSA without the Chromatographic step, recovery of E₂17β added to plasma was only possible if the step was included.

The cross-reactivity of estrone (E₁)with E₂17β using anti-E₂17β-17-BSA as defined by Abraham (J. Clin. Endocr. , 866 (1969) was examined under conditions of constant and of changing E₁:E₂17β ratio.     

βB1-crystallin: thermodynamic profiles of molecular interactions

Background

β-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both βA3 and βB2 crystallins (βA3 and βB2) involves endothermic enthalpy of association (∼8 kcal/mol) mediated by hydrophobic interactions.

Methodology/Principal Findings

Thermodynamic profiles of the associations of dimeric βA3 and βB1 and tetrameric βB1/βA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of βB1 crystallin are dominated by exothermic enthalpy (−13.3 and −24.5 kcal/mol, respectively).

Conclusions/Significance

Global thermodynamics of βB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those β-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations.     

PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching

Recently, it has been shown that PKA-mediated phosphorylation of the β₂-adrenergic receptor (β₂-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G_s and increases its affinity for G_i. Here we demonstrate that, like the β₂-AR, the β₁-AR is also capable of “switching” its coupling from G_s to G_i in a PKA-dependent manner. The β₁-AR is capable of activating adenylate cyclase via G_s, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β₁-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G_i/G_o, and to the PKA inhibitor, H-89. β₁-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G_s-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β₁-AR, like the β₂-AR, can undergo PKA-dependent “G_s/G_i switching”.     

Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of beta-arrestins

β-Arrestins play a role in AT₁ endocytosis by binding the cytoplasmic, C-terminus region T332–S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting β-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT₁ to investigate two possibilities: activated AT₁ induces global relocation of β-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract β-arrestins. Results obtained using high osmolarity and dominant-negative β-arrestin confirmed that internalization of AT₁ in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving β-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT₁ internalization. Confocal microscopy revealed that wild-type AT₁ induced a time-dependent translocation of GFP-tagged β-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic β-arrestin 1 at all, and only trafficked β-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both β-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and β-arrestin 1 less so than β-arrestin 2. In conclusion, this study shows that the high affinity binding of β-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced β-arrestin recruitment to the cell surface and AT₁ receptor.     

Six new C21 steroidal glycosides from Asclepias curassavica L

Six new C₂₁ steroidal glycosides, named curassavosides A–F (3–8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (4), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (5), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (8), respectively. All compounds (1–8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.     

The binding of eosin-labeled subunit δ to the isolated chloroplast ATPase, CF1, as revealed by rotational diffusion in solution

We investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5-isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF₁ (containing δ) and of CF₁(−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2b = 100 Å/2a = 28 Å. Binding of δ to CF₁ decreased the rotational correlation time about 10-fold. The result was a biphasic decay of the laser flash-induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF₁) relative to δ (free). CF₁(−δ), which completely lacked the δ-subunit, bound one δ (mol/mol) with high affinity (K_d ≈ 100 nM) and at least another δ with about 20-fold lower affinity. The δ-containing CF₁, revealed only the low-affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF₁.     

稳定表达人源GABAAR-CHO细胞株的建立

目的: 构建α₁亚基诱导表达、β₂和γ_2L亚基稳定表达的人源α₁β₂γ_2L-GABA_AR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α₁、β₂、γ_2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α₁β₂γ_2L-GABA_AR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α₁、β₂、γ_2L亚基,构建的α₁β₂γ_2L-GABA_AR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α₁亚基并与β₂、γ_2L组装成具有功能活性的α₁β₂γ_2L-GABA_AR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α₁β₂γ_2L-GABA_AR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α₁β₂γ_2L-GABA_AR的激动效应;在膜电位检测研究中,获得GABA激动效应EC₅₀为(177.72 ± 15.92)nmol/L,Dia变构效应EC₅₀为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC₅₀为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α₁β₂γ_2L-GABA_AR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。