Ethanol Increases GABAA Responses in Cells Stably Transfected with Receptor Subunits

Ethanol enhancement of GABA_A receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., ³⁸Cl^- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk^-) cells stably transfected with α₁+β or α+β₁+γ_2L GABA_A receptor subunit DNAs and compared ³⁸Cl flux with whole-cell, patch-clamp measurements of GABA_A receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the -γ-subunit and was maximal at a concentration of 10 m m . Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing α₁β_1-γ_2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 m m . Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABA_A receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a y-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.     

beta(2)-Adrenergic receptor and adenylate cyclase gene polymorphisms affect sickle red cell adhesion

Sickle red cell (SS RBC) adhesion is thought to contribute to sickle cell disease (SCD) pathophysiology. SS RBC adhesion to laminin increases in response to adrenaline stimulation of β₂-adrenergic receptors (β₂ARs) and adenylate cyclase (ADCY6), and previous evidence suggests such activation occurs in vivo . We explored whether polymorphisms of the β₂AR and ADCY6 genes ( ADRB2 and ADCY6 , respectively) affect RBC adhesion to laminin. We found that the β₂AR arg¹⁶→gly substitution and two non-coding ADCY6 polymorphisms were associated with elevated adhesion. We postulate that ADRB2 and ADCY6 polymorphisms may influence SCD severity through the mechanism of RBC adhesion.     

Effects of Ethanol on Recombinant Glycine Receptors Expressed in Mammalian Cell Lines

We examined the effects of acute ethanol exposure on recombinant human glycine receptors transiently transfected into HEK 293 cells and stably transfected into Ltk⁻ fibroblast-like cells. In our study of the effects of ethanol, we used the whole-cell patch-clamp configuration. Relatively low concentrations of ethanol (25 mM and 50 mM) did not affect glycine-gated currents in any of the cell lines studied. Higher concentrations of ethanol (100 mM and 200 mM) significantly potentiated glycine responses only in stably transfected Ltk⁻ cells expressing α₁ and α₂ subunits and in HEK 293 cells transiently expressing α₂ subunits. Cells stably expressing α₁ versus α₂ glycine receptors were modulated equally by ethanol. Both glycine α₁ and glycine α₁β receptors transiently expressed in HEK 293 cells were insensitive to all concentrations of ethanol tested; however, there was a trend toward potentiation at 100 and 200 mM ethanol concentrations. A population of cells (41–87%) that was sensitive to the potentiating effects of 100 and 200 mM ethanol (defined as more than 10% potentiation) was identified in both cell lines tested. In these sensitive cells, ethanol (100 and 200 mM) produced significant potentiation, independent of the cell line and the glycine receptor sub-unit tested. Together with published results from studies with Xenopus oocytes, these data indicate that the sensitivity of recombinant glycine receptors to ethanol depends upon the expression system.     

β2 glycoprotein-I antigen is increased in primary hyperlipidaemia

Summary. In order to determine whether elevated levels of β₂ glycoprotein-I (β₂GPI) are associated with increased plasma lipids, we measured plasma β₂ GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean β₂GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/1. respectively) compared to controls (199.6 mg/1) (p<0.01). Significant correlations were demonstrated between β₂GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r =0.559: P<0.5), but were not observed in all patient groups. These results indicate that β₂GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therfore be considered when interpreting results of β₂GPI antigen assays.     

Realities of Newer β-Blockers for the Management of Hypertension

β-blockers are prescribed for a variety of cardiovascular conditions including hypertension, heart failure, primary treatment of myocardial infarction (MI), and secondary prevention of ischemic cardiac events. Yet they remain underprescribed in populations at increased risk for cardiovascular disease because of tolerability and safety concerns. β-Blockers are heterogeneous with respect to pharmacokinetic and pharmacodynamic effects. "Original" agents were nonselective, blocking both β₁-adrenoceptors and β₂-adrenoceptors. Later, new agents were developed with selectivity for β₁-adrenoceptors, and were subsequently followed by β-blockers, which exhibit additional effects, such as vasodilation. Among newer agents, labetalol, carvedilol, and nebivolol have been approved for use in the United States. Nebivolol possesses both β₁-selectivity and nitric oxide–mediated vasodilatory effects, while carvedilol has attractive effects on insulin resistance and exhibits antioxidant effects. Newer β-blockers may overcome concerns about efficacy, adverse effects, and tolerability, while delivering cardiovascular protection.     

The Binding of Vitamin B12 by Serum Proteins in Normal and B12-Deficient Subjects

Endogenous B₁₂ in normal serum has been shown to be associated with α globulins (Pitney, Beard and Van Loon, 1954; Heinrich and Erdmann-Oehlecker, 1956; Mendelsohn, Watkin, Horbett and Fahey, 1958). When B₁₂ has been added to normal serum, however, most or all of the added B₁₂ has been associated with the β- and α₂-globulins (Miller, 1958). In the present investigation with radioactive ₅₇cobalt cyanocobalamin (B₁₂*) added to serum, it was possible to define at least two B₁₂-binding proteins (BP), a β-globulin, and an α₁ globulin. B₁₂* added to the serum of normal subjects migrated mainly with the β-globulins on electrophoresis, but when added to the serum of B₁₂-deficient subjects, a small fraction of the B₁₂* was also associated with the α₂-globulins.     

Biochemical Studies of a New Class of Alcohol Dehydrogenase Inhibitors from Radix puerariae

Two potent, reversible inhibitors of human alcohol dehydrogenase; (ADH) isozymes were isolated from Radix puerariae (RP, commonly, known as kudzu root) and identified as the isoflavones daidzein and genistein. The 4-methoxy derivatives of daidzein (trivial name, for-mononetin) and genistein (biochanin A), minor constituents of RP, were also shown to be ADH inhibitors. All of these isoflavones inhibit 1 the human -γ₁γ₂-ADH isozyme competitively with respect to ethanol] and uncompetitively with respect to NAD⁺. A survey of more than 40 structurally related compounds revealed one more isoflavone (prunetin) and four flavones (7-hydroxyflavone, apigenin, galangin, and kaempferol) that inhibit ADH. The isoflavone inhibitors, however, are far more potent than the flavone inhibitors. Among the isoflavones studied, genistein is the most potent with K_i , = 0.1 μM toward γ₂γ₂- ADH. Human ADH isozymes differ in their sensitivity to these inhibitors in the order γ₂γ₂- , γ₁γ₁ > α₁α₁⁻,>⁻ > xx-ADH. These inhibitors, do not affect the β₁β₁ ,⁻ and β₂β₂ -ADH isozymes at concentrations as high as 20 μM. Rat and rabbit class I ADHs are also inhibited by these. isoflavone inhibitors. The 7-O-glucosyl derivatives of daidzein, genistein, formononetin, and biochanin A do not inhibit ADH, but are potent aldehyde dehydrogenase inhibitors.     

Serologic and Immunochemical Characterization of Ax Blood

Serologic and immunochemical analyses of A_x bloods have defined a probable specific antibody to the A_x antigen in group O serum. Anti-A_x activity was found to be in 19S (β_2M, γ_1A) and β_2A (γ_1A) globulin fractions but not in the 7S (γ₂) globulin of human group O sera.

Résumé

Les analyses immuno-chimiques et sérologiques des sangs A_x ont permis de déceler un anticorps spécifique contre l'antigène A_x dans les sérums de sang de groupe O. L'activité anti-A_x a été trouvée dans les fractions de globulines 19S (β_2M, γ_1A) et β_2A (γ_1A) mais pas dans la fraction des globulines 7S (γ₂) des sérums des personnes de groupe O.

Zusammenfassung

Anläßlich der serologischen und immunochemischen Analyse von A_x-Blutproben gelang es zu zeigen, daß im O-Serum offenbar ein spezifischer Antikörper gegen das A_x-Antigen enthalten ist. Die Anti-A_x-Aktivität fand sich in den 19S (β_2M, γ_1A) und β_2A (γ_1A) Globulinfraktionen, nicht aber in der 7S (γ₂) Globulinfraktion der menschlichen O-Seren.     

The use of an anti-β2-glycoprotein-I assay for discrimination between anticardiolipin antibodies associated with infection and increased risk of thrombosis

Summary. Antiphospholipid antibodies (aPAs), occurring in association with infection, are not generally associated with an increased risk of thrombosis. Anticardiolipin antibodies (aCL) from patients with infection, unlike those from patients with SLE, do not have the β₂GPI cofactor requirements. Antibodies to β₂GPI (αβ₂GPI) are more closely associated with a previous history of thrombosis than aCL in patients with SLE. In the present study we have investigated the reactivity of the αβ₂GPI assay for aPAs associated with infection. Serum from 114 patients with infections including syphilis ( n = 11), tuberculosis ( n = 63) and Klebsiella ( n =42) were assayed for αβ₂GPI and aCL antibodies. The incidence of aCL in serum of patients with tuberculosis, Klebsiella infection and syphilis was 6.0%. 5.0% and 64.0%. respectively, but all patients were negative for αβ₂GPI. These results indicate that the αβ₂GPI assay is negative in patients with transiently positive aCL assays associated with infection.     

Anti β2 glycoprotein I antibodies: detection and association with thrombosis

Summary. It has been demonstrated that antiphospholipid antibodies (aPL) recognize epitopes formed by anionic phospholipids and protein cofactors. β₂ glycoprotein I (β₂GPI) is accepted as the cofactor of anticardiolipin antibodies (aCL). In the present study we explored the presence and clinical associations of anti β₂GPI antibodies of IgG isotype (aβ₂GPI-IgG), measured by ELISA. We studied sera from 169 patients with aCL and/or lupus anticoagulant (LA), including 52 patients with systemic lupus erythematosus and 49 with primary antiphospholipid syndrome (PAPS). We found 31.9% positive sera for aβGPI-IgG in the whole population and 48.6% in the aCL-IgG(+) group. There was a good correlation between the titre of aCL-IgG and the optical density for aβGPI-IgG ( r = 0.69, P <0.01). The presence of aβ₂GPI-IgG was associated with the presence of aCL-IgG ( P <0.0001) and LA ( P <0.0005). However, none of 23 LA (+) patients without aCL had aβ₂GPI-IgG.
We found a statistically significant association between the presence of aβ₂GPI-IgG and a history of venous thromboembolism (VTE) in our patients ( P <0.005). This association was observed in PAPS ( P <0.05) but not in secondary antiphospholipid syndrome (SAPS).
Our study confirms that some aPL(+) sera react with β₂GPI in special experimental conditions. In addition, the presence of these antibodies is associated with a history of VTE.     

BK channel subunit composition modulates molecular tolerance to ethanol

Background:  The large conductance calcium-activated potassium channel (also called BK channel or Slo channels) is a well-studied target of alcohol action, and plays an important role in behavioral tolerance.
Methods:  Using patch clamp electrophysiology, we examined human BK channels expressed in HEK293 cells to test whether tolerance to ethanol occurs in excised patches and whether it is influenced by subunit composition. Three combinations were examined: hSlo, hSlo + β₁, and hSlo + β₄.
Results:  The 2 components of BK alcohol adaptation (Component 1: rapid tolerance to acute potentiation, and Component 2: a more slowly developing decrease in current density) were observed, and varied according to subunit combination. Using a 2-exposure protocol, Component 1 tolerance was evident in 2 of the 3 combinations, because it was more pronounced for hSlo and hSlo + β₄.
Conclusions:  Thus, rapid tolerance in human BK occurs in cell-free membrane patches, independent of cytosolic second messengers, nucleotides or changes in free calcium. Alcohol pretreatment for 24 hours altered subsequent short-term plasticity of hSlo + β₄ channels, suggesting a relationship between classes of tolerance. Finally, Component 2 reduction in current density showed a striking dependency on channel composition. Twenty-four hour exposure to 25 mM ethanol resulted in a down-regulation of BK current in hSlo and hSlo + β₄ channels, but not in hSlo + β₁ channels. The fact that hSlo + β₁ channels show less sensitivity to acute challenge, in conjunction with less Component 1 and Component 2 tolerance, suggests subunit composition is an important factor for these elements of alcohol response.     

GM-CSF RAISES SERUM LEVELS OF β2-MICROGLOBULIN AND THYMIDINE KINASE IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKAEMIA

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on biochemical tumour markers β₂-microglobulin (β₂m), thymidine kinase (TK) and lactate dehydrogenase (LDH) were studied in eight patients with chronic lymphocytic leukaemia (CLL). The serum concentration of β₂m rose by a median of 30% (range 8–50%) and serum TK by 101% (range 30–1414%). Serum LDH concentration, on the other hand, significantly decreased in all patients. The significant increases of β₂m and TK could not be explained by progression of the disease or impaired renal function. Treatment with GM-CSF reduces the value of serum β₂m and TK in assessment of tumour mass and disease activity.     

The β+ IVS, I-NT no. 6 (T→C) thalassaemia in heterozygotes with an associated Hb Valletta or Hb S heterozygosity in homozygotes from Malta

Summary. In vitro DNA amplification and dot blot analysis with synthetic allele specific oligonucleotides (ASO) identified the β IVS, I-6 (T→C) thalassaemia in 78% of 32 chromosomes from 16 β-thalassaemia homozygotes in Malta. The preponderance of a single thalassaemia mutation in one population is unusual. The β⁺ IVS, I-6C thalassaemia mutation was also found in three carriers who had an associated β globin heterozygosity, i.e. Hb Valletta (or α₂β₂^87PRO) or Hb S (or α₂β₂^6VAL). The proportion of Hb A in these cases (av. = 29.7%) provided objective documentation of the relatively mild effect of this mutation on in vivo globin gene expression. However, the expression of homozygous disease was more severe in developing children compared to adults. The β⁺ IVS, I-6C mutation complicates population testing because heterozygotes can have Hb A₂ levels below those classically associated with β thalassaemia.     

Pregnancy Associated Plasma Protein-A: Interaction with Heparin in Crossed Affinity lmmunoelectrophoresis

Abstract: A specific interaction between pregnancy associated plasma protein-A (PAPP-A) arid heparin has been demonstrated using heparin affinity crossed immuno electrophoresis applied to late pregnancy serum. The presence of heparin in the first dimensional gel accelerated the anodic migration of six serum proteins with β electrophoretic mobility and one protein in the α mobile region. Two of the six β mobile proteins were identified as anti-thrombin III and β lipoprotein, the α mobile protein being PAPP-A. The migration distance of other proteins originating in the placenta (pregnancy specific β₁ glycoprotein), fetus (alpha-fetoprotein). leucocytes (pregnancy zone protein), or maternal liver (α₁ antitrypsin α₂ macroglobulin) was not altered. The interaction of PAPP-A with heparin was therefore independent of Molecular size, charge, and site of origin indicating a specific high affinity interaction between PAPP-A and heparin. These results indicate that PAPP-A may be involved locally with the coagulation system in the maintenance of placental circulation .     

Beta 3-adrenergic receptor gene polymorphism and type 2 diabetes in a Caucasian population

Summary
Aim   The β₃-adrenergic receptor (β₃-AR) is suspected to play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. A mutation in the β₃-AR gene (Trp64Arg) has been associated with the capacity of weight gain and with early onset of noninsulin dependent diabetes mellitus (type 2 diabetes). In this study we investigated the prevalence of the two β₃-AR alleles in a Caucasian population and studied the association between the β₃-AR genotype and metabolic disorders (obesity and type 2 diabetes).
Methods   Genomic DNA extracted from peripheral blood leucocytes of 200 Caucasian subjects (137 subjects with and 63 subjects without type 2 diabetes). The Mva I polymorphism of β₃-AR, which detects the Trp64Arg mutation, was determined by polymerase chain reaction (PCR). We studied the correlation between the Trp64Arg mutation and the body mass index (b.m.i. kg/m²).
Results   There was no significant difference between the patients with type 2 diabetes and control subjects in the frequency of the Arg64 allele (5.5% and 4.8%, respectively). Within the group of type 2 diabetes patients were 14 subjects with the Trp64Arg mutation (b.m.i., mean ± s.d.: 31 ± 8.5 kg/m²) and 123 without the mutation (b.m.i. 29 ± 4.8). There was no association between the β₃-AR gene polymorphism and sex, obesity, blood pressure, glycohaemoglobin concentration, proteinuria.
Conclusion   Our results suggest that the Trp64Arg mutation is not a major determinant of metabolic disorders (type 2 diabetes, obesity) and chronic complications of type 2 diabetes in a Dutch population.     

Congenital Methaemoglobinaemia due to NADH Methaemoglobin Reductase Deficiency: Successful Treatment with Oral Riboflavin

S ummary . A Japanese family with congenital methaemoglobinaemia is described. The family pedigree was compatible with autosomal recessive type of inheritance. The increased methaemoglobin concentration was ascribed to the red cell NADH diaphorase deficiency associated with the almost complete lack of one of the two peaks of the diaphorase activity as separated by DEAE Sephadex column chromato-graphy. The NADH diaphorase and NADH methaemoglobin reductase deficiency was limited to the red cells. The methaemoglobin content in the blood of the propositus was 17.8% and isoelectric focusing analysis on a polyacrylamide gel plate showed that the haemoglobin consisted of 65.2% oxyhaemoglobin (α²⁺β²⁺)₂, 29.6% half-oxidized forms, 20.9% (α+β²+)₂ and 8.7% (α²⁺β⁺)₂, and 3% full-oxidized methaemoglobin (α⁺β⁺)₂. Oral administration of riboflavin 120 mg/d resulted in a gradual but significant decrease in the level of the met-form haemoglobins in parallel with a gradual increase in the red cell flavin content. Riboflavin is considered to be effective by activating the NADPH diaphorase (NADPH flavin reductase) system and appears to be useful for the treatment of congenital methaemoglobinaemia.     

Compartmentalization regulates the interaction between the platelet integrin αIIbβ3 and ICln

The volume-regulating protein, ICln, interacts with the conserved KxGFFKR α-integrin signature motif. ICln is an abundant protein (4455 ± 650 molecules/platelet) found exclusively in the soluble cytosolic fraction of unactivated platelets. In contrast, its binding partner, the platelet integrin α_IIbβ₃, is present in detergent-insoluble fractions associated with membrane and cytoskeleton subcellular localizations. This study investigated factors that regulate the interaction of ICln with α_IIbβ₃ during platelet activation. His-tagged recombinant ICln bound equally to purified α_IIbβ₃ and to integrin from resting or activated platelets. Binding was not affected by direct integrin activation with Mn⁺⁺ or by inhibitors of integrin occupancy (abciximab, RGD). However, the capacity for interaction between integrin and recombinant ICln was slowly downregulated following prolonged platelet activation for >300 s. In parallel, ICln redistributed to membrane and cytoskeletal platelet subcellular fractions. The time-course of this redistribution preceded the downregulation of integrin binding capacity and suggests that only a short window of opportunity exists for ICln interaction with α_IIbβ₃ to occur. Thus, although ICln has the inherent capacity to bind to α_IIbβ₃ regardless of its activation state, it can only do so following platelet activation. Activation-dependent subcellular redistribution of ICln represents a novel, temporally-regulated mechanism for control of integrin function in platelets.     

Allotypes of Serum α2-Macroglobulin β-Lipoprotein and Immunoglobulins in the Domesticated Sheep (Ovis Aries)

Abstract. Antisera specific for allotypic markers carried by the serum α₂-macro-globulin, β-lipoprotein and IgGl and IgG2 were produced during cross-immunization experiments with sheep (Ovis aries). Two markers controlled by autosomal co-dominant genes, Ap ¹ and Ap ², were found on the α₂-macroglobulin; one marker controlled by an autosomal dominant Lp ¹ was found on the β-lipoprotein, two markers controlled by the autosomal codominants, Igl ¹ and Igl ², were found on the IgGl and one marker, controlled by Ig2 ¹ and linked to Igl ¹, was found on the IgG2. The immunoglobulin markers all occurred on the Fc fragments of the immunoglobulin molecules. There was no linkage between the Ap and Lp loci or between these and the immunoglobulin loci. A preliminary survey of the incidences of the markers in various flocks of sheep showed that the incidence of Lp ¹ and Igl ¹ was very much higher in Merinos than in the British breeds of sheep.