Bile salts of germ-free domestic fowl and pigs

1. The bile of germ-free domestic fowl contains taurine conjugates of 3α,7α-dihydroxy-5β-cholan-24-oic acid (chenodeoxycholic acid), 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (cholic acid) and its 5α-epimer (allocholic acid): that of germ-free pigs contains glycine and taurine conjugates of chenodeoxycholic acid, 3α,6α-dihydroxy-5β-cholan-24-oic acid (hyodeoxycholic acid), 3α,6α,7α-trihydroxy-5β-cholan-24-oic acid (hyocholic acid) and (probably) cholic acid. Keto acids were not found. 2. Allocholic acid and hyodeoxycholic acid are thus proved to be primary bile acids in intact animals. 3. The evolutionary and biochemical implications of these findings are briefly considered.     

Formation of Hyodeoxycholic Acid from Muricholic Acid and Hyocholic Acid by an Unidentified Gram-Positive Rod Termed HDCA-1 Isolated from Rat Intestinal Microflora

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3α,6β,7β-trihydroxy bile acid β-muricholic acid into hyodeoxycholic acid (3α,6α-dihydroxy acid) by dehydroxylation of the 7β-hydroxy group and epimerization of the 6β-hydroxy group into a 6α-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3α,6α,7α-trihydroxy acid), α-muricholic acid (3α,6β,7α-trihydroxy acid), and ω-muricholic acid (3α,6α,7β-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted β-muricholic acid almost completely into hyodeoxycholic acid.     

Properties of beta-lactamases produced by three species of mycobacteria

1. Mycobacterium smegmatis (N.C.T.C. 8158), M. fortuitum and M. phlei (MPI) produce a constitutive β-lactamase that has penicillinase and cephalosporinase activity. 2. The β-lactamases of these three species of acid-fast bacteria were mainly cell-bound, only small amounts of activity being liberated into the extracellular fluid. The total β-lactamase activity of these mycobacteria was much lower than that of certain Gram-positive organisms, but comparable with that reported for species of Gram-negative bacteria. 3. The β-lactamases of intact cells of the mycobacteria were not freely accessible to any of the substrates tested, but the apparent crypticity factor to benzylpenicillin was greater than that to cephaloridine and cephalosporin C. 4. Attempts to induce β-lactamase activity in M. smegmatis and M. phlei failed even with high concentrations of inducer. 5. The β-lactamases obtained from the three species of mycobacteria showed different substrate specificities, including different relative activities as cephalosporinases and penicillinases respectively. 6. Certain derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid were found to be resistant to hydrolysis by β-lactamases of M. smegmatis and M. fortuitum. 7. The β-lactamase of M. smegmatis was competitively inhibited by a number of β-lactamase-resistant derivatives of 6-aminopenicillanic acid, but not by similar derivatives of 7-aminocephalosporanic acid.     

Tomato 14-3-3 Protein 7 Positively Regulates Immunity-Associated Programmed Cell Death by Enhancing Protein Abundance and Signaling Ability of MAPKKK α

Programmed cell death (PCD) is triggered when Pto, a Ser-Thr protein kinase, recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv tomato. This PCD requires mitogen-activated protein kinase kinase kinase (MAPKKK α ) as a positive regulator in tomato (Solanum lycopersicum) and Nicotiana benthamiana. To examine how PCD-eliciting activity of the tomato MAPKKK α protein is regulated, we screened for MAPKKK α -interacting proteins in tomato and identified a 14-3-3 protein, TFT7. Virus-induced gene silencing using the TFT7 gene in N. benthamiana compromised both Pto- and MAPKKK α -mediated PCD, and coexpression of TFT7 with tomato MAPKKK α enhanced MAPKKK α -mediated PCD. TFT7 was also required for PCD associated with several other disease resistance proteins and contributed to resistance against P. syringae pv tomato. Coexpression of TFT7 with MAPKKK α in vivo caused increased accumulation of the kinase and enhanced phosphorylation of two MAP kinases. TFT7 protein contains a phosphopeptide binding motif that is present in human 14-3-3 ϵ, and substitutions in this motif abolished interaction with MAPKKK α in vivo and also the PCD-enhancing activity of TFT7. A 14-3-3 binding motif, including a putative phosphorylated Ser-535, is present in the C-terminal region of MAPKKK α. An S535A substitution in MAPKKK α reduced interaction with TFT7 and both PCD-eliciting ability and stability of MAPKKK α. Our results provide new insights into a role for 14-3-3 proteins in regulating immunity-associated PCD pathways in plants.     

Estrogen Receptor-α36 Is Involved in Pterostilbene-Induced Apoptosis and Anti-Proliferation in In Vitro and In Vivo Breast Cancer

Pterostilbene (trans-3,5-dimethoxy-4′-hudroxystilbene) is an antioxidant primarily found in blueberries. It also inhibits breast cancer regardless of conventional estrogen receptor (ER-α66) status by inducing both caspase-dependent and caspase-independent apoptosis. However, the pterostilbene-induced apoptosis rate in ER-α66-negative breast cancer cells is much higher than that in ER-α66-positive breast cancer cells. ER-α36, a variant of ER-α66, is widely expressed in ER-α66-negative breast cancer, and its high expression mediates the resistance of ER-α66-positive breast cancer patients to tamoxifen therapy. The aim of the present study is to determine the relationship between the antiproliferation activity of pterostilbene and ER-α36 expression in breast cancer cells. Methyl-thiazolyl-tetrazolium (MTT) assay, apoptosis analysis, and an orthotropic xenograft mouse model were used to examine the effects of pterostilbene on breast cancer cells. The expressions of ER-α36 and caspase 3, the activation of ERK and Akt were also studied through RT-PCR, western blot analysis, and immunohistochemical (IHC) staining. ER-α36 knockdown was found to desensitize ER-α66-negative breast cancer cells to pterostilbene treatment both in vitro and in vivo, and high ER-α36 expression promotes pterostilbene-induced apoptosis in breast cancer cells. Western blot analysis data indicate that MAPK/ERK and PI3K/Akt signaling in breast cancer cells with high ER-α36 expression are mediated by ER-α36, and are inhibited by pterostilbene. These results suggest that ER-α36 is a therapeutic target in ER-α36-positive breast cancer, and pterostilbene is an inhibitor that targets ER-α36 in the personalized therapy against ER-α36-positive breast cancer.     

Comparative studies of bile salts. Bile salts of the lamprey Petromyzon marinus L

1. Bile salts of Petromyzon marinus L. ammocoetes appeared to consist solely or chiefly of a crystalline substance, whose chromatographic and i.r.-spectral characteristics suggested that it was a monosulphate ester of a bile alcohol having the 3α,7α,12α-trihydroxy pattern of substitution in a 5α-steroid nucleus. 2. This substance on cleavage with dioxan–trichloroacetic acid gave petromyzonol, n.m.r. and mass-spectral examination of which suggested the structure 5α-cholane-3α,7α,12α,24-tetrol. 3. 3α,7α,12α-Trihydroxy-5α-cholanoic acid (allocholic acid) from the lizards Anolis lineatopus lineatopus Gray and Cyclura carinata Harlan (family Iguanidae) was esterified with propan-1-ol and reduced by lithium aluminium hydride to 5α-cholane-3α,7α,12α,24-tetrol, identical with petromyzonol. 4. Chromic acid oxidation of petromyzonol sulphate from lamprey bile, followed by acid hydrolysis, gave 24-hydroxy-5α-cholane-3,7,12-trione; hence the sulphate ester group is at C-24. 5. Petromyzonol sulphate is both primitive and unique: a study of its biogenesis might improve our understanding of evolution at the molecular level.     

Chemical Chaperones Exceed the Chaperone Effects of RIC-3 in Promoting Assembly of Functional α7 AChRs

Functional α7 nicotinic acetylcholine receptors (AChRs) do not assemble efficiently in cells transfected with α7 subunits unless the cells are also transfected with the chaperone protein RIC-3. Despite the presence of RIC-3, large amounts of these subunits remain improperly assembled. Thus, additional chaperone proteins are probably required for efficient assembly of α7 AChRs. Cholinergic ligands can act as pharmacological chaperones to promote assembly of mature AChRs and upregulate the amount of functional AChRs. In addition, we have found that the chemical chaperones 4-phenylbutyric acid (PBA) and valproic acid (VPA) greatly increase the amount of functional α7 AChRs produced in a cell line expressing both α7 and RIC-3. Increased α7 AChR expression allows assay of drug action using a membrane potential-sensitive fluorescent indicator. Both PBA and VPA also increase α7 expression in the SH-SY5Y neuroblastoma cell line that endogenously expresses α7 AChRs. VPA increases expression of endogenous α7 AChRs in hippocampal neurons but PBA does not. RIC-3 is insufficient for optimal assembly of α7 AChRs, but provides assay conditions for detecting additional chaperones. Chemical chaperones are a useful pragmatic approach to express high levels of human α7 AChRs for drug selection and characterization and possibly to increase α7 expression in vivo.     

Susceptibility of Salmonellae to Cephalosporins and to Nine Other Antimicrobial Agents

Three cephalosporin-related antibiotics and nine other antimicrobial agents were studied for in vitro effectiveness against 54 recently isolated strains of Salmonella. Minimal inhibitory concentrations determined by the plate dilution method demonstrated the following percentages of resistance: ampicillin, 6%; tetracycline, 13%; streptomycin, 52%; sulfadiazine, 94%; cephaloglycin, 96%; and lincomycin, 100%. No strains were resistant to cephalothin, cephaloridine, chloramphenicol, colistimethate, kanamycin, and polymyxin B. The commonest serotype studied, S. typhimurium, showed the greatest antibiotic resistance, with 21% resistant to ampicillin, 36% resistant to tetracycline, and 71% resistant to streptomycin. Cephalothin and cephaloridine were highly effective in vitro but inhibitory concentrations of 20 to 40 mug of cephaloglycin per ml were required for the majority of Salmonella strains.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

Survey of Tall-Fescue Pasture: Correlation of Toxicity of Fusarium Isolates to Known Toxins

Several aspects of fescue foot in cattle suggest that this disease is caused by fungi growing on fescue grass. Certain fungi isolated from winter pasture yield toxins when grown on synthetic medium. Most of these toxin producers belong to the genus Fusarium. All but 1 of the 21 toxic and 7 questionably toxic Fusarium isolates produce either 4-acetamido-4-hydroxy-2-butenoic acid γ-lactone, or 4β, 15-diacetoxy-8α-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3α-ol, or both.     

In Plant Protoplasts, the Spontaneous Expression of Defense Reactions and the Responsiveness to Exogenous Elicitors Are under Auxin Control

When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of β-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of β-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating β-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced β-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 × 10⁻⁵ molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco β-glucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.     

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR₉₂-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase K_m for α-ketoisovaleric acid, 6 × 10⁻⁵ M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K_m for α-ketoisovaleric acid, 1.1 × 10⁻³ M; transaminase B K_m for α-ketoisovaleric acid, 2 × 10⁻³ M).     

Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

Adiponectin, an adipocyte-derived insulin-sensitizing and anti-inflammatory hormone, is suppressed in obesity through mechanisms involving chronic inflammation and oxidative stress. Olive oil consumption is associated with beneficial cardiometabolic actions, with possible contributions from the antioxidant phenol hydroxytyrosol (HT) and the monounsaturated fatty acid oleic acid (OA, 18:1n-9 cis), both possessing anti-inflammatory and vasculo-protective properties. We determined the effects of HT and OA, alone and in combination, on adiponectin expression in human and murine adipocytes under pro-inflammatory conditions induced by the cytokine tumor necrosis factor(TNF)-α. We used human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes and murine 3T3-L1 adipocytes as cell model systems, and pretreated them with 1-100 μmol/L OA, 0.1-20 μmol/L HT or OA plus HT combination before stimulation with 10 ng/mL TNF-α. OA or HT significantly (P<0.05) prevented TNF-α-induced suppression of total adiponectin secretion (by 42% compared with TNF-α alone) as well as mRNA levels (by 30% compared with TNF-α alone). HT and OA also prevented—by 35%—TNF-α-induced downregulation of peroxisome proliferator-activated receptor PPARγ. Co-treatment with HT and OA restored adiponectin and PPARγ expression in an additive manner compared with single treatments. Exploring the activation of JNK, which is crucial for both adiponectin and PPARγ suppression by TNF-α, we found that HT and OA additively attenuated TNF-α-stimulated JNK phosphorylation (up to 55% inhibition). In conclusion, the virgin olive oil components OA and HT, at nutritionally relevant concentrations, have additive effects in preventing adiponectin downregulation in inflamed adipocytes through an attenuation of JNK-mediated PPARγ suppression.     

An electrophoretic investigation of groundnut proteins: the structure of arachins A and B

1. The proteins of the groundnut cotyledon have been fractionated and analysed by DEAE-Sephadex chromatography and acrylamide-gel electrophoresis. Seventeen components were detected. 2. A new method is described for the preparation of arachin, using calcium precipitation. The product contains at least 99% of arachin. 3. The theory of acrylamide-gel electrophoresis is developed and applied to the arachin system to predict the molecular weight of one sub-unit of arachin. 4. A variant form of arachin, arachin B, has been discovered. Of 81 nuts, 27 contained only arachin B, 53 contained both arachin A and B, and one contained arachin A only. This is almost certainly a polymorphism of arachin; this is the first example of polymorphism to be reported in plant proteins. 5. A combination of controlled denaturation, electrophoretic analysis, ultracentrifuge and Sephadex filtration data has shown that arachin A contains four different kinds of peptide chains (α, β, γ and δ). Arachin B contains only β, γ and δ chains. 6. The most probable structure for arachin B, mol.wt. 330000 form, is 8 β, 2 γ and 2 δ chains, and for arachin A, 4 α, 4 β, 2 γ and 2 δ chains. Arachin without β chains was not found. 7. The α and β chains have mol.wts. of about 35000 and the γ and δ chains of about 10000. 8. Three N-terminal groups were found: the α and β chains both terminate in glycine; the γ and δ chains terminate in isoleucine and glutamic acid. 9. Arachin contains no carbohydrate. 10. Disulphide bonds are not important in arachin: there are none between the α, β, γ and δ chains. 11. The amino acid compositions of arachins A and B are very similar. Glutamic acid and aspartic acid residues are exceptionally frequent.     

Distant Homology Modeling of LCAT and Its Validation through In Silico Targeting and In Vitro and In Vivo Assays

LCAT (lecithin:cholesterol acyltransferase) catalyzes the transacylation of a fatty acid of lecithin to cholesterol, generating a cholesteryl ester and lysolecithin. The knowledge of LCAT atomic structure and the identification of the amino acids relevant in controlling its structure and function are expected to be very helpful to understand the enzyme catalytic mechanism, as involved in HDL cholesterol metabolism. However - after an early report in the late ‘90 s - no recent advance has been made about LCAT three-dimensional structure. In this paper, we propose an LCAT atomistic model, built following the most up-to-date molecular modeling approaches, and exploiting newly solved crystallographic structures. LCAT shows the typical folding of the α/β hydrolase superfamily, and its topology is characterized by a combination of α-helices covering a central 7-strand β-sheet. LCAT presents a Ser/Asp/His catalytic triad with a peculiar geometry, which is shared with such other enzyme classes as lipases, proteases and esterases. Our proposed model was validated through different approaches. We evaluated the impact on LCAT structure of some point mutations close to the enzyme active site (Lys218Asn, Thr274Ala, Thr274Ile) and explained, at a molecular level, their phenotypic effects. Furthermore, we devised some LCAT modulators either designed through a de novo strategy or identified through a virtual high-throughput screening pipeline. The tested compounds were proven to be potent inhibitors of the enzyme activity.     

Anti-inflammatory effects of antibacterials on human bronchial epithelial cells

Background

Human Bronchial epithelial cells (hu-BEC) have been claimed to play a significant role in the pathogenesis of chronic inflammatory airway diseases like COPD. In this context IL-8 and GM-CSF have been shown to be key cytokines. Some antibiotics which are routinely used to treat lower respiratory tract infections have been shown to exert additional immunomodulatory or anti-inflammatory effects. We investigated whether these effects can also be detected in hu-BEC.

Methods

Hu-BEC obtained from patients undergoing lung resections were transferred to air-liquid-interface (ALI) culture. These cultures were incubated with cefuroxime (CXM, 10-62.5 mg/l), azithromycin (AZM, 0.1-1.5 mg/l), levofloxacin (LVX, 1-8 mg/l) and moxifloxacin (MXF, 1-16 mg/l). The spontaneous and TNF-α (10 ng/ml) induced expression and release of IL-8 and GM-CSF were measured using PCR and ELISA in the absence or presence of these antibiotics.

Results

The spontaneous IL-8 and GM-CSF release was significantly reduced with MXF (8 mg/l) by 37 ± 20% and 45 ± 31%, respectively (both p < 0.01). IL-8 release in TNF-α stimulated hu-BEC decreased by 16 ± 8% (p < 0.05) with AZM (1.5 mg/l). With MXF a concentration dependent decrease of IL-8 release was noted up to 39 ± 7% (p < 0.05). GM-CSF release from TNF-α stimulated hu-BEC was maximally decreased by 35 ± 24% (p < 0.01) with MXF (4 mg/l).

Conclusion

Using ALI cultures of hu-BEC we observed differential effects of antibiotics on spontaneous and TNF-α induced cytokine release. Our data suggest that MXF and AZM, beyond bactericidal effects, may attenuate the inflammatory process mediated by hu-BEC.     

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp²³⁰ residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.     

α7 Nicotinic Acetylcholine Receptor-Specific Antibody Induces Inflammation and Amyloid β42 Accumulation in the Mouse Brain to Impair Memory

Nicotinic acetylcholine receptors (nAChRs) expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ₄₂), memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208) or injected with bacterial lipopolysaccharide (LPS) for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ₄₀ and Aβ₄₂ and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208) resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ₄₂ and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1) neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ₄₂ accumulation and memory impairment in mice and (2) α7(1-208) nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.