The α5 Subunit Regulates the Expression and Function of α4*-Containing Neuronal Nicotinic Acetylcholine Receptors in the Ventral-Tegmental Area

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.     

A PCR-based method to test for the presence or absence of β-conglycinin α′- and α-subunits in soybean

The soybean cultivar Yumeminori, which lacks the α′- and α-subunits of β-conglycinin, carries both naturally occurring and induced mutations. While the cause of the natural mutation resulting in the α′-subunit deficiency has been determined, the induced mutation in the CG-2 gene encoding the α-subunit has not been characterized at the molecular level. In this study, we identified a four base pair insertion in the first exon of CG-2, which introduced a premature stop codon. The insertion co-segregated with the lack of α-subunit, indicating that this mutation is the cause of the α-subunit deficiency. A multiplex PCR method of testing for the presence or absence of α′- and α-subunits was developed based on the sequences of mutated and wild-type alleles. This PCR-based test was also capable of detecting the presence of wild-type genes when Yumeminori DNA samples were contaminated with wild-type DNA at levels of 0.2% or greater. Thus, this method will be useful both for marker-assisted selection in soybean breeding programs, and for seed purity tests in food industries.     

The Genetics of α-Hydroxyacid Oxidase and Alcohol Dehydrogenase in the Mouse: Evidence for Multiple Gene Loci and Linkage between Hao-2 and Adh-3

Electrophoretic polymorphisms for stomach alcohol dehydrogenase (ADH-C2) and kidney L-alpha-hydroxyacid oxidase (HAOX-B4) have been identified in an Asian subspecies of mouse, Musmusculus castaneous. These variants are inherited in a normal Mendelian fashion with two alleles in each case showing codominant expression. The structural gene loci for those enzymes (Adh-3 and Hao-2, respectively) are apparently linked (17.6% recombinants) in this organism, whereas the multiple gene loci for HAOX, Hao-1 (encoding the A4 liver isozyme) and Hao-2, exhibited independent segregation and are unlinked (50% recombinants). Evidence is presented for 3 ADH loci: ADH-1, encoding liver ADH-A2 which exhibits high activity with ethanol (SELANDER, HUNT and YANG 1969; ADH-2, liver and stomach ADH-B2 using 2-hexene-1-ol as substrate; and Adh-3, stomach ADH-C2 using both benzyl alcohol and 2-hexene-1-ol as substrate.     

Molecular Docking and Dynamic Simulation to Identify α7nAChR Binding Affinity of Flavonoids for the Treatment of Alzheimer's Disease

Background : Alpha-7-nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel is one of the important parts of the cholinergic pathway in the brain and has a remarkable role in Alzheimer's disease (AD). It has been documented that the modulation of α7nAChR with the help of phytoconstituent can be helpful in the treatment of AD. Method : The binding efficacy of fifty flavonoids was evaluated for human α7nAChR using molecular docking. The best two flavonoids shortlisted from docking analysis were then subjected to molecular dynamic simulations for 100 ns to analyze conformational binding stability with the target protein. Further, the druggability of the selected flavonoids was checked using in silico ADMET studies. Result : The top two flavonoids selected based on binding affinity toward the binding site of α7nAChR from molecular docking were amentoflavone (–9.1 kcal/mol) and gallocatechin (–8.8 kcal/mol). The molecular dynamics simulation revealed that amentoflavone and gallocatechin have a stable state during overall simulation time, lesser root mean deviation (RMSD) and root mean square fluctuation (RMSF), and complex of both compounds with protein is stable until 100 ns. Conclusion : The two flavonoids amentoflavone and gallocatechin are potential lead molecules that could be utilized as effective agonists of α7nAChR to combat Alzheimer's disease. Future in vitro and in vivo analyses are required to confirm their effectiveness.     

The Roles of Helix I and Strand 5A in the Folding,Function and Misfolding of α1-Antitrypsin

α₁-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α₁-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α₁-Antitrypsin has never been examined. We have therefore created a range of α₁Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α₁-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states.     

Alcohol Selectivity of β3-Containing GABAA Receptors: Evidence for a Unique Extracellular Alcohol/Imidazobenzodiazepine Ro15-4513 Binding Site at the α+β- Subunit Interface in αβ3δ GABAA Receptors

GABA_A receptors (GABARs) have long been the focus for acute alcohol actions with evidence for behaviorally relevant low millimolar alcohol actions on tonic GABA currents and extrasynaptic α4/6, δ, and β3 subunit-containing GABARs. Using recombinant expression in oocytes combined with two electrode voltage clamp, we show with chimeric β2/β3 subunits that differences in alcohol sensitivity among β subunits are determined by the extracellular N-terminal part of the protein. Furthermore, by using point mutations, we show that the β3 alcohol selectivity is determined by a single amino acid residue in the N-terminus that differs between GABAR β subunits (β3Y66, β2A66, β1S66). The β3Y66 residue is located in a region called “loop D” which in γ subunits contributes to the imidazobenzodiazepine (iBZ) binding site at the classical α+γ2- subunit interface. In structural homology models β3Y66 is the equivalent of γ2T81 which is one of three critical residues lining the benzodiazepine binding site in the γ2 subunit loop D, opposite to the “100H/R-site” benzodiazepine binding residue in GABAR α subunits. We have shown that the α6R100Q mutation at this site leads to increased alcohol-induced motor in-coordination in alcohol non-tolerant rats carrying the α6R100Q mutated allele. Based on the identification of these two amino acid residues α6R100 and β66 we propose a model in which β3 and δ containing GABA receptors contain a unique ethanol site at the α4/6+β3- subunit interface. This site is homologous to the classical benzodiazepine binding site and we propose that it not only binds ethanol at relevant concentrations (EC₅₀–17 mM), but also has high affinity for a few selected benzodiazepine site ligands including alcohol antagonistic iBZs (Ro15-4513, RY023, RY024, RY80) which have in common a large moiety at the C7 position of the benzodiazepine ring. We suggest that large moieties at the C7-BZ ring compete with alcohol for its binding pocket at a α4/6+β3- EtOH/Ro15-4513 site. This model reconciles many years of alcohol research on GABARs and provides a plausible explanation for the competitive relationship between ethanol and iBZ alcohol antagonists in which bulky moieties at the C7 position compete with ethanol for its binding site. We conclude with a critical discussion to suggest that much of the controversy surrounding this issue might be due to fundamental species differences in alcohol and alcohol antagonist responses in rats and mice.     

A comparison of the in vitro binding of α-tocopherol to microsomes of lung,liver, heart and brain of the rat

The in vitro binding of α-tocopherol to microsomes of lung, liver, heart and brain of the rat was studied with the insoluble tocopherol ligand presented as a complex with bovine serum albumin. Under these conditions, all microsomes showed nonsaturable binding of α-tocopherol and the amount bound to microsomes was linearly proportional to the concentration of albumin-complexed tocopherol. Increasing the amount of α-tocopherol bound to microsomes in this manner reduced the extent of lipid peroxidation induced by added ferrous iron. The apparent affinities of the microsomes for α-tocopherol, as indicated by the amount bound at a given concentration of albumin-complexed tocopherol, decreased in the order brain > liver ≈ heart > lung. The differences in affinity did not correlate with total fatty acid content (r = − 0.39), total unsaturated fatty acid content (r = − 0.26), or with the content of fatty acids containing two or more double bonds (r = − 0.01). A high positive correlation was found with the content of fatty acids containing three or more double bonds (r = + 0.96). Since lung microsomes contain approx. 6-times the tocopherol levels of liver and brain and about twice that of heart microsomes, these results show that the in vivo levels of microsomal tocopherol do not reflect microsomal affinity for this biological antioxidant.     

Pharmacophore models based studies on the affinity and selectivity toward 5-HT1A with reference to α1-adrenergic receptors among arylpiperazine derivatives of phenytoin

The study is focused on (2-alkoxy)phenylpiperazine derivatives of 1-(2-hydroxy-3-(4-arylpiperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione with alkyl or ester substituents at N3 of hydantoin ring, as well as a new designed and synthesized series of compounds with a free N3H group or N3-acetic acid terminal fragment. The compounds were assessed on their affinity for 5-HT_1A and ??₁-adrenoceptors and evaluated in functional bioassays for antagonistic properties. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) and DFT methods (B3LYP functional, Gaussian 0.3) were used to investigate 3D structure of the compounds. SAR analysis was based on two pharmacophore models, the one described by Barbaro et al. for ??₁-adenoceptor antagonist and the model of Lepailleur et al. for 5-HT_1A receptor ligands. All compounds exhibited significant to moderate affinities for 5-HT_1A receptors in nanomolar range (7-610 nM). The highest activity (7 nM) and selectivity (17.38) for 5-HT_1A was observed for 1-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (13a). Among new synthesized compounds 1-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride (20a) displayed the highest affinity (16.6 nM) and selectivity (5.72) for ??₁-AR.     

Synthesis and in vitro binding studies of piperazine-alkyl-naphthamides: Impact of homology and sulphonamide/carboxamide bioisosteric replacement on the affinity for 5-HT1A, α2A,D4.2, D3 and D2L receptors

A series of carboxamide and sulphonamide alkyl(ethyl to hexyl)piperazine analogues were prepared and tested for their affinity to bind to a range of receptors potentially involved in psychiatric disorders. These chemical modifications led us to explore the impact of homology and bioisosteric replacement of the amide group. All of these compounds possessed a high affinity for 5-HT_1A receptors, irrespective of the size of the linker, the carboxamide derivative with a pentyl linker had the highest affinity for α_2A receptor sites and also a high affinity for 5-HT_1A and D3 receptors. The sulphonamide analogue with a hexyl linker possessed a high affinity for 5-HT_1A, D4.2 and D3 receptors.     

Identification of Three Residues Essential for 5-Hydroxytryptamine 2A-Metabotropic Glutamate 2 (5-HT2A·mGlu2) Receptor Heteromerization and Its Psychoactive Behavioral Function

Serotonin and glutamate G protein-coupled receptor (GPCR) neurotransmission affects cognition and perception in humans and rodents. GPCRs are capable of forming heteromeric complexes that differentially alter cell signaling, but the role of this structural arrangement in modulating behavior remains unknown. Here, we identified three residues located at the intracellular end of transmembrane domain four that are necessary for the metabotropic glutamate 2 (mGlu2) receptor to be assembled as a GPCR heteromer with the serotonin 5-hydroxytryptamine 2A (5-HT_2A) receptor in the mouse frontal cortex. Substitution of these residues (Ala-677^4.40, Ala-681^4.44, and Ala-685^4.48) leads to absence of 5-HT_2A·mGlu2 receptor complex formation, an effect that is associated with a decrease in their heteromeric ligand binding interaction. Disruption of heteromeric expression with mGlu2 attenuates the psychosis-like effects induced in mice by hallucinogenic 5-HT_2A agonists. Furthermore, the ligand binding interaction between the components of the 5-HT_2A·mGlu2 receptor heterocomplex is up-regulated in the frontal cortex of schizophrenic subjects as compared with controls. Together, these findings provide structural evidence for the unique behavioral function of a GPCR heteromer.     

Activation of 5-HT1A receptors in medullary raphé disrupts sleep and decreases shivering during cooling in the conscious piglet

Activation of 5-HT1A receptors in the medullary raphé decreases sympathetically mediated brown adipose tissue (BAT) thermogenesis and peripheral vasoconstriction when previously activated with leptin, LPS, prostaglandins, or cooling. It is not known whether shivering is also modulated by medullary raphé 5-HT1A receptors. We previously showed in conscious piglets that activation of 5-HT1A receptors with (+/-)-8-hydroxy-2-(dipropylamino)-tetralin (8-OH-DPAT) in the paragigantocellularis lateralis (PGCL), a medullary region lateral to the raphé that contains substantial numbers of 5-HT neurons, eliminates rapid eye movement (REM) sleep and decreases shivering in a cold environment, but does not attenuate peripheral vasoconstriction. Hoffman JM, Brown JW, Sirlin EA, Benoit AM, Gill WH, Harris MB, Darnall RA. Am J Physiol Regul Integr Comp Physiol 293: R518-R527, 2007. We hypothesized that, during cooling, activation of 5-HT1A receptors in the medullary raphé would also eliminate REM sleep and, in contrast to activation of 5-HT1A receptors in the PGCL, would attenuate both shivering and peripheral vasoconstriction. In a continuously cool environment, dialysis of 8-OH-DPAT into the medullary raphé resulted in alternating brief periods of non-REM sleep and wakefulness and eliminated REM sleep, as observed when 8-OH-DPAT is dialyzed into the PGCL. Moreover, both shivering and peripheral vasoconstriction were significantly attenuated after 8-OH-DPAT dialysis into the medullary raphé. The effects of 8-OH-DPAT were prevented after dialysis of the selective 5-HT1A receptor antagonist WAY-100635. We conclude that, during cooling, exogenous activation of 5-HT1A receptors in the medullary raphé decreases both shivering and peripheral vasoconstriction. Our data are consistent with the hypothesis that neurons expressing 5-HT1A receptors in the medullary raphé facilitate spinal motor circuits involved in shivering, as well as sympathetic stimulation of other thermoregulatory effector mechanisms.     

Regulating Prefrontal Cortex Activation: An Emerging Role for the 5-HT2A Serotonin Receptor in the Modulation of Emotion-Based Actions?

The prefrontal cortex (PFC) is involved in mediating important higher-order cognitive processes such as decision making, prompting thereby our actions. At the same time, PFC activation is strongly influenced by emotional reactions through its functional interaction with the amygdala and the striatal circuitry, areas involved in emotion and reward processing. The PFC, however, is able to modulate amygdala reactivity via a feedback loop to this area. A role for serotonin in adjusting for this circuitry of cognitive regulation of emotion has long been suggested based primarily on the positive pharmacological effect of elevating serotonin levels in anxiety regulation. Recent animal and human functional magnetic resonance studies have pointed to a specific involvement of the 5-hydroxytryptamine (5-HT)_2A serotonin receptor in the PFC feedback regulatory projection onto the amygdala. This receptor is highly expressed in the prefrontal cortex areas, playing an important role in modulating cortical activity and neural oscillations (brain waves). This makes it an interesting potential pharmacological target for the treatment of neuropsychiatric modes characterized by lack of inhibitory control of emotion-based actions, such as addiction and other impulse-related behaviors. In this review, we give an overview of the 5-HT_2A receptor distribution (neuronal, intracellular, and anatomical) along with its functional and physiological effect on PFC activation, and how that relates to more recent findings of a regulatory effect of the PFC on the emotional control of our actions.     

The structure of the ends of α-helices in globular proteins: effect of additional hydrogen bonds and implications for helix formation

We prepared a set of about 2000 α-helices from a relational database of high-resolution three-dimensional structures of globular proteins, and identified additional main chain i ← i+3 hydrogen bonds at the ends of the helices (i.e., where the hydrogen bonding potential is not fulfilled by canonical i ← i+4 hydrogen bonds). About one-third of α-helices have such additional hydrogen bonds at the N-terminus, and more than half do so at the C-terminus. Although many of these additional hydrogen bonds at the C-terminus are associated with Schellman loops, the majority are not. We compared the dihedral angles at the termini of α-helices having or lacking the additional hydrogen bonds. Significant differences were found, especially at the C-terminus, where the dihedral angles at positions C2 and C1 in the absence of additional hydrogen bonds deviate substantially from those occurring within the α-helix. Using a novel approach we show how the structure of the C-terminus of the α-helix can emerge from that of constituent overlapping α-turns and β-turns, which individually show a variation in dihedral angles at different positions. We have also considered the direction of propagation of the α-helix using this approach. If one assumes that helices start as a single α-turn and grow by successive addition of further α-turns, the paths for growth in the N → C and C → N directions differ in a way that suggests that extension in the C → N direction is favored.     

Development of a Highly Sensitive Immuno-PCR Assay for the Measurement of α-Galactosidase A Protein Levels in Serum and Plasma

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.     

Distinct Structural Requirements for Interaction of the Integrins α5β1, αvβ5, and αvβ6 with the Central Cell Binding Domain in Fibronectin

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these, α5β1, αIIbβ3, and αvβ3, the structural requirements for binding to fibronectin differ. In the present study. we report that two additional integrins, αvβ6. and αvβ5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments. αvβ5, like αvβ3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study. αvβ5 only minimally supported adhesion to intact fibronectin. but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat. alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated by αvβ5, as well as adhesion mediated by αvβ6. αvβ5 and αvβ6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.     

Exploration of computational approaches to predict the structural features and recent trends in α-amylase production for industrial applications

Amylases are biologically active enzymes that can hydrolyze starch to produce dextrin, glucose, maltose, and oligosaccharides. The amylases contribute approximately 30% to the global industrial enzyme market. The globally produced amylases are widely used in textile, biofuel, starch processing, food, bioremediation of environmental pollutants, pulp, and paper, clinical, and fermentation industries. The purpose of this review article is to summarize recent trends and aspects of α-amylases, classification, microbial production sources, biosynthesis and production methods, and its broad-spectrum applications for industrial purposes, which will depict the latest trends in α-amylases production. In the present article, we have comprehensively compared the biodiversity of α-amylases in different model organisms ranging from archaea to eukaryotes using in silico structural analysis tools. The detailed comparative analysis: regarding their structure, function, cofactor, signal peptide, and catalytic domain along with their catalytic residues of α-amylases in 16 model organisms were discussed in this paper. The comparative studies on alpha (α) amylases' secondary and tertiary structures, multiple sequence alignment, transmembrane helices, physiochemical properties, and their phylogenetic analysis in model organisms were briefly studied. This review has documented the recent trends and future perspectives of industrially important novel thermophilic α-amylases. In conclusion, this review sheds light on the current understanding and prospects of α-amylase research, highlighting its importance as a versatile enzyme with numerous applications and emphasizing the need for further exploration and innovation in this field.