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.     

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.     

Cytoskeletal Changes in the Brains of Mice Lacking Calcineurin Aα

Abstract: Hyperphosphorylated τ, the major component of the paired helical filaments of Alzheimer's disease, was found to accumulate in the brains of mice in which the calcineurin Aα gene was disrupted [calcineurin Aα knockout (CNAα^−/−)]. The hyperphosphorylation involved several sites on τ, especially the Ser³⁹⁶ and/or Ser⁴⁰⁴ recognized by the PHF-1 monoclonal antibody. The increase in phosphorylated τ content occurred primarily in the mossy fibers of the CNAα^−/− hippocampus, which contained the highest level of calcineurin in brains of wild-type mice. The CNAα^−/− mossy fibers also contained less neurofilament protein than normal, although the overall level of neurofilament phosphorylation was unchanged. In the electron microscope, the mossy fibers of CNAα^−/− mice exhibited abnormalities in their cytoskeleton and a lower neurofilament/microtubule ratio than those of wild-type animals. These findings indicate that hyperphosphorylated τ can accumulate in vivo as a result of reduced calcineurin activity and is accompanied by cytoskeletal changes that are likely to have functional consequences on the affected neurons. The CNAα^−/− mice were found in a separate study to have deficits in learning and memory that may result in part from the cytoskeletal changes in the hippocampus.     

Production and properties of ααα-glucosidase from the thermotolerant bacteriumThermomonospora curvata

Thermomonospora curvata contains α-1,4-glucosidase that is induced duringgrowth on maltose and starch. Maltose acts as an inducer of α-glucosidase even in thepresence of glucose. An intracellular thermostable α-glucosidase from T. curvata wasdetected in the crude extract on SDS-PAGE by means of modified colour reaction afterrenaturation of the enzyme. The enzyme was purified 59-fold to homogeneity with a yield of17·7% by a combination of ion-exchange and hydrophobic interaction chromatography andgel filtration. The enzyme has an apparent molecular mass of 60±1 kDa and isoelectric point4·1. The α-glucosidase exhibits optimum activity at pH 7·0–7·5 and54°C. The activity is inhibited by heavy metals and is positively affected by Ca²+ andMg²+. The enzyme hydrolyses maltose, sucrose, p-nitrophenyl-α- d -glucopyranoside and maltodextrins from maltotriose up to maltoheptaose with a decreasingefficiency. The K_m for maltose and p-NPG are 12 and 2·3 mmol l⁻¹,respectively.     

Changes in the Expression of the αα Form of Enolase During Neuroblastoma Differentiation

Abstract: The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form αα, and an increase in the activity associated with the γ-containing isozymes (αγ plus γγ); in the absence of DMSO, there is no decrease in αα or in total enolase activity. In order to study the mechanism of the changes in αα, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the α antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the α antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the α antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t_1/2 > 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the α subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de-expression of the α gene that occurs in vivo during neuronal differentiation.     

Role of the N-terminal α-helix in biogenesis of α7 nicotinic receptors

We studied the role of the α-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in α7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the α-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of α7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (α3β4 and α4β2) and 5-HT_3A receptors also abolished their expression at the membrane. We conclude that the N-terminal α-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.     

Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABA_A receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABA_A receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α₁ and α₅ subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α₁ and α₅ subunit immunoreactivity was reduced. In the VTA, levels of α₁ subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α₁ subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α₅ mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABA_A receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.     

Structural Analysis of the Sixth Immunoglobulin-Like Domain of Mouse Neural Cell Adhesion Molecule L1 and Its Interactions with αvβ3, αIIbβ3, and α5β1 Integrins

Abstract: Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to αvβ3, αIIbβ3, and α5β1 integrins. The domain was found to have the expected β-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to αvβ3, αIIbβ3, and α5β1 integrins, suggesting specific interactions with these ligands. However, only the interaction to αvβ3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC₅₀ was determined to be ∼20 n M . Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562–564 is involved in the interaction to αvβ3. Our findings indicate an RGD-independent mechanism for the interactions to αIIbβ3 and α5β1, as no involvement of any RGD motif could be demonstrated.     

3α-Hydroxysteroid Oxidoreductase in Rat Brain

Abstract: We describe a simple procedure for the microassay of 3α-hydroxysteroid oxidoreductase in homogenates of rat brain. This enzyme converts dihydrotestosterone to 3α-androstandiol. We have mapped the distribution of the enzymatic activity in 14 regions of the rat brain. The highest activities were observed in homogenates of olfactory bulb (51/nmol/mg protein/h) and olfactory tubercle (29 nmol/mg protein/h). Substantially lower values were seen in the other brain regions, including thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, and preoptic area (6–20 nmol/mg protein/ h).     

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.     

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.     

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.     

β-adducin (Add2) KO mice show synaptic plasticity, motor coordination and behavioral deficits accompanied by changes in the expression and phosphorylation levels of the α- and γ-adducin subunits

Adducins are a family of proteins found in cytoskeleton junctional complexes, which bind and regulate actin filaments and actin-spectrin complexes. In brain, adducin is expressed at high levels and is identified as a constituent of synaptic structures, such as dendritic spines and growth cones of neurons. Adducin-induced changes in dendritic spines are involved in activity-dependent synaptic plasticity processes associated with learning and memory, but the mechanisms underlying these functions remain to be elucidated. Here, β-adducin knockout (KO) mice were used to obtain a deeper insight into the role of adducin in these processes. We showed that β-adducin KO mice showed behavioral, motor coordination and learning deficits together with an altered expression and/or phosphorylation levels of α-adducin and γ-adducin. We found that β-adducin KO mice exhibited deficits in learning and motor performances associated with an impairment of long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. These effects were accompanied by a decrease in phosphorylation of adducin, a reduction in α-adducin expression levels and upregulation of γ-adducin in hippocampus, cerebellum and neocortex of mutant mice. In addition, we found that the mRNA encoding β-adducin is also located in dendrites, where it may participate in the fine modulation of LTP and LTD. These results strongly suggest coordinated expression and phosphorylation of adducin subunits as a key mechanism underlying synaptic plasticity, motor coordination performance and learning behaviors.     

Localization of Na,K-ATPase α/β Isoforms in Rat Sciatic Nerves : Effect of Diabetes and Fish Oil Treatment

The localization of the Na,K-ATPase isoenzymes in sciatic nerve remains controversial, as well as diabetes-induced changes in Na,K-ATPase isoforms. Some of these changes could be prevented by fish oil therapy. The aim of this study was to determine by confocal microscopy the distribution of Na,K-ATPase isoforms (alpha1, alpha2, alpha3, beta1, and beta2) in the sciatic nerve, the changes induced by diabetes, and the preventive effect of fish oil in diabetic neuropathy. This study was performed in three groups of rats. In the first two groups, diabetes was induced by streptozotocin and rats were supplemented daily with fish oil or olive oil at a dosage of 0.5 g/kg of body weight. The third one was a control group that was supplemented with olive oil. Five antibodies against specific epitopes of Na,K-ATPase isoenzymes were applied to stained dissociated nerve fibers with fluorescent secondary antibodies. The five isoenzymes were documented in nonspecific regions, Schwann cells (myelin), and the node of Ranvier. The localization of the alpha1, alpha2, and beta1 isoenzymes was not affected by diabetes. In contrast, diabetes induced a decrease of the alpha2 subunit (p < 0.05) and an up-regulation of the beta2 subunit (p < 0.05). These modifications were noted in both regions for alpha2 and were localized at the myelin domain only for the beta2. Fish oil supplementation prevented the diabetes-induced changes in the alpha2 subunit with an additional up-regulation. The beta2 subunit was not modified. A phenotypic change similar to nerve injury was induced by diabetes. Fish oil supplementation partially prevented some of these changes.     

Partial purification of cell wall α-galactosidases and α-arabinosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

Two protein fractions with activity as α-galactosidase (EC 3.2.1.22) and α-arabinosidase (EC 3.2.1.55), respectively, were identified in the proteins of cell wall of Cicer arietinum L. cv. Castellana extracted with 3 M LiCl. These fractions were partially purified by gel filtration chromatography (Bio Gel P-150), increasing the specific arabinosidase activity 57-fold and the α-galactosidase activity 6-fold. Other protein fractions with glucosidase (EC 3.2.1.21) and glucanase (EC 3.2.1.6) activity also appeared. According to earlier authors, α-arabinosidases and α-galactosidases are related to alterations in linkages occurring in cell walls, since the enzymes are able to hydrolyze isolated wall polymers. However, our preparations hydrolyze intact cell walls only to a very limited extent, such that their participation in the autolytic processes of cell walls can be ruled out.     

Immunohistochemical Distribution of RGS7 Protein and Cellular Selectivity in Colocalizing with Gαq Proteins in the Adult Rat Brain

Abstract : Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins αi/o and αq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Gαq/11, an important G protein-coupled receptor signal transducer for phospholipase Cβ-mediated activity. In general, both RGS7 and Gαq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Gαq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Gαq/11-mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Gαq/11. We also describe a possible RGS7-selective neuronal feedback adaptation on Gαq/11-mediated pathway function, which may play an important role in signaling specificity in the brain.     

Alterations of Na,K-ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n-3 Fatty Acids

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase α subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1- and α3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.     

Involvement of Glycoproteins in the Development of Early Mouse Embryos: Effect of Tunicamycin and α, α' Dipyridyl in vitro

Early mouse embryos grown in tissue culture were treated with tunicamycin, an inhibitor of protein glycosylation or with αα' dipyridyl, an inhibitor of collagen secretion. Neither treatment blocked development of cleavage stage embryos nor did either interfere with blastocyst formation, hatching, or adhesion to the substratum at low concentrations. However, both treatments caused marked and specific changes in the morphology of the blastocyst outgrowth. Treatment of embryos with tunicamycin caused severe deterioration of the trophoblast layer and subsequent disintegration of the inner cell mass. Tunicamycin completely inhibited the incorporation of mannose into proteins. Treatment with αα' dipyridyl caused dose dependent retardation of the inner cell mass while the trophoblast cells were virtually unaffected. These alterations in morphogenesis occurred only in embryos treated at the blastocyst stage or later in development. Changes caused by α,α' dipyridyl could be partially reversed by addition of collagen to the culture. These findings might indicate the involvement of extracellular matrix macromolecules in embryonic organization.