Platelet activation by a relapsing fever spirochaete results in enhanced bacterium–platelet interaction via integrin αIIbβ3 activation

Borrelia hermsii , a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro . Platelet binding was promoted by the platelet integrin α_IIbβ₃: the bacterium bound to purified integrin α_IIbβ₃, and bacterial binding to platelets was diminished by α_IIbβ₃ antagonists or by a genetic defect in this integrin. Integrin α_IIbβ₃ undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of α_IIbβ₃ that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin α_IIbβ₃ activation, as indicated by the expression of the platelet activation marker P-selectin and integrin α_IIbβ₃ in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I₂, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.     

α2-Macroglobulin Attenuates β-Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons

Abstract: β-Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α₂-macroglobulin (α₂M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α₂M is an Aβ binding protein. We now report that, in contrast to another plaque-associated protein, α₁-antichymotrypsin, α₂M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α₂M. We postulate that α₂M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.     

Rat Frontal Cortex β1-Adrenoceptors Are Activated by the β3-Adrenoceptor Agonists SR 58611A and SR 58878A but Not by BRL 37344 or ICI 215,001

Abstract: SR 58611A, a selective agonist of gut and brown adipose tissue β₃-adrenoceptors (β₃ARs), has been reported to have antidepressant-like activity in rodents by indicating brain β₃ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine- or (−)-isoproterenol-stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by β₁AR antagonist CGP 20712A but not by β₂AR antagonist ICI 118,551. In addition, PCR analysis did not reveal β₃AR mRNA, and no specific β₃AR binding sites were detected by [³H]CGP 12177 in rat frontal cortex. When down-regulation of the β₁AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (−)-isoproterenol increased the cyclic AMP production less markedly. Our findings indicate that β₃ARs are absent in the adult rat frontal cortex, and that various β₃AR agonists differently affect the frontal cortex β₁ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex β₁ARs.     

α2-Macroglobulin Complexes with and Mediates the Endocytosis of β-Amyloid Peptide via Cell Surface Low-Density Lipoprotein Receptor-Related Protein

Abstract: A primary histopathological feature of Alzheimer's disease is the accumulation of β-amyloid (Aβ) in the brain of afflicted individuals. However, Aβ is produced continuously as a soluble protein in healthy individuals where it is detected in serum and CSF, suggesting the existence of cellular clearance mechanisms that normally prevent its accumulation and aggregation. Here, we demonstrate that Aβ forms stable complexes with activated α₂-macroglobulin (α₂M^⋆), a physiological ligand for the low-density lipoprotein receptor-related protein (LRP) that is abundantly expressed in the CNS. These α₂M^⋆/¹²⁵I-Aβ complexes are immunoreactive with both anti-Aβ and anti-α₂M IgG and are stable under various pH conditions, sodium dodecyl sulfate, reducing agents, and boiling. We demonstrate that α₂M^⋆/¹²⁵I-Aβ complexes can be degraded by glioblastoma cells and fibroblasts via LRP, because degradation is partially inhibited by receptor-associated protein (RAP), an antagonist of ligand interactions with LRP. In contrast, the degradation of free ¹²⁵I-Aβ is not inhibited by RAP and thus must be mediated via an LRP-independent pathway. These results suggest that LRP can function as a clearance receptor for Aβ via a physiological ligand.     

Developmental Characterization of Thymosin β4 and β10 Expression in Enriched Neuronal Cultures from Rat Cerebella

Abstract: The β₄ and β₁₀ thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin β₄ is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin β₁₀ is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the β-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both β-thymosins were initially expressed in granule cells, although expression, especially of thymosin β₄, was truncated compared with the in vivo time course. As in vivo, thymosin β₄ was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin β₁₀. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the β-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating β-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.     

A Calcium Channel from the Presynaptic Nerve Terminal of the Narke japonica Electric Organ Contains a Non-N-Type α2δ Subunit

Abstract: A monoclonal antibody (MCC-1) that recognizes the α₂δ subunit complex of L-type calcium channels from rabbit skeletal muscle membranes partially inhibited the evoked release of acetylcholine from synaptosomes isolated from the electric organ of the marine electric ray, Narke japonica . Digitonin extracts of synaptosomal plasma membranes were subjected to immunoaffinity column chromatography on MCC-1-Sepharose. The purified fraction contained a 170-kDa protein that reacts with MCC-1 and dissociates into smaller polypeptides under reducing conditions. In addition, immunoblotting analysis revealed the existence of syntaxin in the purified fraction, suggesting that the calcium channel forms a complex with syntaxin. However, MCC-1 did not immunoprecipitate an ω-conotoxin GVIA-binding protein. These findings indicate that the 170-kDa protein may be the α₂δ subunit of a calcium channel that is distinct from the ω-conotoxin GVIA-sensitive N-type calcium channel and partially responsible for the calcium influx that triggers the evoked release of acetylcholine.     

Functional Rescue of β1-Adrenoceptor Dimerization and Trafficking by Pharmacological Chaperones

Site-directed mutagenesis guided by evolutionary trace analysis revealed that substitution of V179 and W183 within a cluster of evolutionarily important residues on the surface of the fourth transmembrane domain of the β₁-adrenergic receptor (β₁AR) significantly reduced the propensity of the receptor to self-assemble into homodimers as assessed by bioluminescence resonance energy transfer in living cells. These results suggest that mutation of V179 and W183 result in conformational changes that reduce homodimerization either directly by interfering with the dimerization interface or indirectly by causing local misfolding that result in reduced self-assembly. However, the mutations did not cause a general misfolding of the β₁AR as they did not prevent heterodimerization with the β₂AR. The homodimerization-compromised mutants were significantly retained in the endoplasmic reticulum (ER) and could not be properly matured and trafficked to the cell surface. Lipophilic β-adrenergic ligands acted as pharmacological chaperones by restoring both dimerization and plasma membrane trafficking of the ER-retained dimerization-compromised β₁AR mutants. These results clearly indicate that homodimerization occurs early in the biosynthetic process in the ER and that pharmacological chaperones can promote both dimerization and cell surface targeting, most likely by stabilizing receptor conformations compatible with the two processes.     

Regulation of α2A-Adrenergic Receptor Expression by Epinephrine in Cultured Astroglia from Rat Brain

Abstract: Epinephrine (Epi) mediates various physiological effects via α_2A-adrenergic receptors (α_2A-ARs). Studies in mice with a point mutation in the gene for α_2A-AR have shown that these receptors are responsible for the centrally mediated depressor effects of α₂-AR agonists. These studies underscore the importance of understanding the basic cellular mechanisms involved in the expression of α_2A-ARs, of which little is known. We use astroglia cultured from the hypothalamus and brainstem of adult Sprague-Dawley rats as a model system in which to study factors that regulate α_2A-AR expression. These cells contain α₂-ARs, which are predominately of the α_2A-AR subtype. Our studies have shown that Epi causes a dose- and time-dependent decrease in steady-state levels of α_2A-AR mRNA and number of α_2A-ARs, effects that are mediated via α₁- and β-adrenergic receptors (α₁-ARs and β-ARs). These effects of Epi on α_2A-AR mRNA and α_2A-AR number are mimicked by activation of protein kinase C or increases in cellular cyclic AMP, which are intracellular messengers activated by α₁-ARs and β-ARs, respectively. Taken together, these results indicate that expression of α_2A-ARs is regulated in a heterologous manner by Epi, via α₁-AR- and β-AR-mediated intracellular pathways.     

In Situ Hybridization Evidence of Differential Modulation by Pentobarbital of GABAA Receptor α1- and β3-Subunit mRNAs

Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABA_A receptor α₁- and β₃-subunit mRNA was conducted using synthetic 3'- end ³⁵S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α₁-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β₃-subunit mRNA were not affected. Dramatically increased levels of GABA_A receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α₁ subunit and in cerebral cortex only for the β₃-subunit. These data provide further support to the structural and pharmacological GABA_A receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.     

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell–cell contact promotes cell-surface expression of PS1/γ-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Aβ production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N-cadherin expression decreased Aβ_42/40 ratio. The effect of N-cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ_42/40 ratio via PS1/N-cadherin interactions.     

Enhancement of pH‐resistant iron‐binding activity in supernatants of rainbow trout blood leucocytes by in vitro treatment with phorbol‐12‐myristate‐13‐acetate

Leucocyte lysates from rainbow trout Oncorhynchus mykiss showed an iron‐binding activity that was retained even if the samples were exposed to an acid pH (4·5). Iron‐binding activity of leucocyte supernatants was enhanced by the presence of 1 μg ml⁻¹ phorbol‐12‐myristate‐13‐acetate in the cell medium.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Quantitative and qualitative differences in C6‐volatile production from the lipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana

Six‐carbon (C₆) volatile products are released from the enzymatic action of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway and form the basis of the "green‐note" flavour characteristic of many consumed plant products. Arabidopsis leaf tissue contains the C₆‐aldehydes hexanal, and trans ‐2‐hexenal as well as the C₆‐alcohols: hexanol, and 3‐hexenol. Interconversion between C₆‐aldehydes and alcohols is thought to proceed through the action of alcohol dehydrogenase (ADH). Using an ADH mutant of Arabidopsis , we have shown that there are large quantitative and qualitative differences in the accumulation of C₆‐volatiles in the absence of ADH activity. The total quantity of LOX‐derived volatiles is greater on a fresh weight basis in the ADH mutant. Qualitatively, hexanol and 3‐hexenol levels are approximately 62% and 51% lower in the mutant, respectively, whereas levels of hexenal are approximately 10‐fold higher. Hexanal accumulation, however, is unaffected in the mutant. The altered profile of LOX‐derived volatiles does not have an effect on the steady‐state levels of mRNA for allene oxide synthase (AOS) or LOX. HPL activity and mRNA quantity, however, are higher in the mutant relative to wild type, suggesting that altered product levels in the mutant affect HPL regulation.     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.