Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

L1/HNK-1 Carbohydrate- and β1 Integrin-Dependent Neural Cell Adhesion to Laminin-1

Abstract: We have shown recently that mouse small cerebellar neurons adhere to a short amino acid sequence of the G2 domain of the laminin α1 chain via the cell surface-expressed HNK-1 carbohydrate. Therefore, we were interested in identifying glycoproteins carrying the HNK-1 carbohydrate at the cell surface of these neurons. Adhesion of small cerebellar neurons to laminin is partially dependent on Ca²⁺, Mn²⁺, and Mg²⁺, indicating the involvement of integrins, which were identified as β1, α3, and α6. They could be shown to bind to laminin by a β1-dependent adhesion mechanism. None of these subunits was found to carry the HNK-1 carbohydrate. HNK-1-immunoreactive glycoproteins were immunoprecipitated and shown to consist of predominantly one molecular species, which was identified as the neural cell recognition molecule L1. L1 was demonstrated to bind in a concentration-dependent and saturating manner to laminin. The binding could be partially inhibited by Fab fragments of monoclonal antibodies against the HNK-1 carbohydrate and against the Ig-like domains of L1. Furthermore, antibodies to the Ig-like domains of L1 and β1 integrin inhibited partially cell adhesion to laminin. Determination of the association of L1, β1 integrin, and the HNK-1 carbohydrate on the cell surface of live cerebellar neurons by antibody-induced patching and copatching revealed HNK-1 to be linked to L1, but less so to β1 integrin. However, only negligible association was found between L1 and β1 integrin. Furthermore, it could be shown that adhesion to laminin is mediated by L1/HNK-1- and β1 integrin-dependent mechanisms that act at least partially independent of each other.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Multiple Determinants of Dihydro-β-Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-β-erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro-β-erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro-β-erythroidine by ∼120-fold. IC₅₀ values for blocking α4β4 and α3β4, responding to EC₂₀ concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µ M , respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro-β-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro-β-erythroidine sensitivity by 4.3-fold.     

β-Synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with α-synuclein

α-synuclein (αS) and β-synuclein (βS) are homologous proteins implicated in Parkinson's disease and related synucleinopathies. While αS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, βS is considered as a potent inhibitor of αS aggregation and toxicity. No mechanism for the neuroprotective role of βS has been described before. Here, we report that similar to αS, βS normally occurs in lipid-associated, soluble oligomers in wild-type (WT) mouse brains. We partially purified βS and αS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate βS from αS and further separate βS monomer from its own oligomers. Importantly, we show that although αS and βS share high degree of similarities, βS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while αS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero-oligomers of αS and βS and suggest that βS expression inhibits PUFA-enhanced αS oligomerization by forming hetero-oligomers up to a quatramer that do not further propagate.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Production of monoclonal antibodies against 3α,20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

Abstract A procedure is described for the production of monoclonal antibodies (mAbs) against 3α,20β-hydroxysteroid dehydrogenase (3α,20β-HSD) from the actinomycete Streptomyces hydrogenans ATCC 19631. Clones which were obtained after fusion of immune cells were screened by solid-phase ELISA and immunoblotting. About 5.2% of the clones secreted immunoglobulins with specificity for 3α,20β-HSD. The purified mAbs were found to belong to subclass IgG₁ and to recognize both the native enzyme as well as its identical subunits which were obtained by SDS denaturation. However, the activity of the tetrameric holoenzyme was only weakly diminished in the presence of these mAbs.     

Effect of interleukin-1α on the growth of Mycobacterium microti within J774A.1 cells

Abstract The effect of mouse recombinant interleukin-1 α on the intracellular growth of Mycobacterium microti in a murine macrophage cell line J774A.1 was investigated. Interleukin-1 α added after infection to the M. microti -infected macrophage monolayers enhanced the growth of M. microti in a concentration-dependent manner and this growth enhancement was abrogated by neutralization of interleukin-1 α with anti-interleukin-1 α antibody. Cyclic adenosine monophosphate level in J774A.1 cells was increased by the addition of interleukin-1 α . Addition of dibutyryl cyclic adenosine monophosphate to infected J774A.1 cells increased the number of intracellular bacteria in a concentration-dependent manner. These results suggest that interleukin-1 α acts as a growth enhancer for intracellular M. microti and the growth enhancing effect of interleukin-1 α may be due to enhanced cellular cyclic adenosine monophosphate level.     

Production of 5α- and 5β-dihydrotestosterone by isolated human axillary bacteria

Abstract The metabolism of testosterone by coryneform bacteria in vitro has been studied. Metabolites identified after derivatization by capillary gas chromatography and further by combined gas chromatography-mass spectrometry were 17β-hydroxy-5α-androstan-3-one and 17β-hydroxy-5β-androstan-3-one. The mass spectral characteristics of the methyl oxime trimethylsilyl ethers of all the 17-hydroxy-androstan-3-one and 3-hydroxy-androstan-17-one isomers are recorded.     

ADRENERGIC α- AND β-RECEPTORS EXPRESSED BY THE SAME CELL TYPE IN PRIMARY CULTURE OF PERINATAL MOUSE BRAIN

Abstract— The β-adrenergic agonist, isoproterenol and the α- and β-adrenergic agonist. NA. raise the intracellular concentration of cyclic AMP in cultures of dissociated perinatal mouse brain. This rise is prevented by a β- but not by an α-adrenergic antagonist. The maximal level of cyclic AMP reached in the presence of isoproterenol is markedly higher than that found after exposure to NA. However, if NA is used along with an α-adrenergic antagonist, cyclic AMP levels as high as those after isoproterenol are measured. Agonists with α-adrenergic activity including NA decrease the response to isoproterenol. The decrease is blocked by α-adrenergic antagonists. From this and additional evidence it is concluded: (1) The increase in the level of cyclic AMP caused by β-adrenergic agonists is due to β-receptor-mediated stimulation of adenylate cyclase; (2) the inhibition of this effect by α-adrenergic agonists is mediated by adrenergic α-receptors; (3) the α- and β-adrenergic receptors are likely to be located on the same cells, probably the most abundant putative glial precursor cells. The simultaneous stimulation of α- and β-adrenergic receptors on the same cell may be of significance in the regulation of the response to NA.     

A novel fluorescent α-conotoxin for the study of α7 nicotinic acetylcholine receptors

Homomeric α7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified α9 subunit can also form functional homopentamers as well as α9α10 heteropentamers. Current fluorescent probes for α7 nicotinic ACh receptors are derived from α-bungarotoxin (α-BgTx). However, α-BgTx also binds to α9* and α1* receptors which are coexpressed with α7 in multiple tissues. We used an analog of α-conotoxin ArIB to develop a highly selective fluorescent probe for α7 receptors. This fluorescent α-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked α7 currents in Xenopus laevis oocytes with an IC₅₀ value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated α-BgTx, Cy3-ArIB[V11L;V16A] did not block α9α10 or α1β1δε receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [¹²⁵I]-α-BgTx binding to mouse hippocampal membranes with a K _i value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXα7R1 cells but not KXα3β2R4, KXα3β4R2, or KXα4β2R2 cells. This labeling could be abolished by pre-treatment with α-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for α7 receptors.     

Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

On the Identification of α- and β-Tubulin Subunits

Abstract: Confusion appears to have arisen in the literature regarding the designation of α-and β-tubulin in polyacrylamide gels. The presence or absence of 8 M-urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M-urea-SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post-translational tyrosylation of protein appears exclusively restricted to α-tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α₂ subunit of tubulin is tyrosylated.     

Three novel plasmid R6K proteins act in concert to distort DNA within the α and β origins of DNA replication

Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (αLIR and βLIR), which are essential elements for the two distally located R6K α and β DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5' end of αLIR, whereas a gene coding for DDP3 is located at the 3' end of βLIR. DDP1 and DDP2 are required for primary DNA distortion within αLIR or βLIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within αLIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site. The possible relevance of these findings to R6K replication is discussed.     

The α- and β-Tubulin Genes of Euplotes octocarinatus

ABSTRACT. The α- and the β-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The α-tubulin gene is located on a 1,587 bp macronuclear DNA molecule and the β-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the α-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most α-tubulin genes, but it supports findings for other hypotrichous ciliates. No evidence for the existence of more than one type of α- and one type of β-tubulin genes could be obtained.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

Gene regulation of α4β2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effects

α4β2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of α4β2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 μM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in hα4β2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in ³[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1β and IL-6. Nicotine suppresses IL-1β and IL-6 expression at least in part by inhibiting NFκB activation. Antagonists dihydro-β-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing α4β2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal α4β2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of α4β2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate α4β2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic α4β2 receptor activation promotes anti-inflammatory effects similar to α7 receptor activation.