Antibodies to the α(1)-Adrenergic Receptor Cause Vascular Impairments in Rat Brain as Demonstrated by Magnetic Resonance Angiography

Background

Circulating agonistic autoantibodies acting at G protein-coupled receptors have been associated with numerous sever pathologies in humans. Antibodies directed predominantly against the α₁-adrenergig receptor were detected in patients suffering from widespread diseases such as hypertension and type 2 diabetes. Their deleterious action has been demonstrated for peripheral organs. We postulate that antibodies to the α₁-adrenergig receptor are relevant pathomolecules in diseases of the central nervous system associated with vascular impairments.

Methodology/Principal Findings

Using a rat model we studied the long-term action of antibodies against the α₁-adrenergig receptor either induced by immunization with a receptor peptide or applied by intravenous injection. The vasculature in the rat brains was investigated by time-of-flight magnetic resonance angiography using a 9.4 Tesla small animal MR imaging system. Visual examination of maximum-intensity-projections (MIPs) of brain angiographs revealed the development of vascular defects in antibody- exposed animals between three and eight months of treatment. Relative vascular areas were derived from representative MIP image sections by grayscale analysis and used to form an index of vascular circulation. Animals exposed to the action of α₁-adrenergig receptor antibodies showed significantly reduced vascular areas (p<0.05). Calculated index values indicated attenuated blood flow in both antibody-treated cohorts compared to their respective controls reaching with (relative units ± standard error, n = 10) 0.839±0.026 versus 0.919±0.026 statistical significance (p<0.05) for peptide-immunized rats.

Conclusion/Significance

We present evidence that antibodies to the α₁-adrenergig receptor cause cerebrovascular impairments in the rat. Our findings suggest the pathological significance of these antibodies in pathologies of the human central nervous system linked to impairments of brain vasculature such as stroke and dementia.     

Susceptibility to persistent breeding-induced endometritis in the mare: relationship to endometrial biopsy score and age, and variations between seasons

The objectives were to: (1) investigate the associations of age and endometrial biopsy score with uterine fluid retention after insemination; and (2) determine if a strict classification of susceptibility to persistent breeding-induced endometritis (PBIE) based on biopsy score, endometrial cytology, and fluid retention after inseminations, is consistent over subsequent breeding seasons. In Experiment 1, 57 mares were inseminated with 10⁹ freeze-killed sperm during estrus and evaluated for uterine fluid retention 48 h and 96 h after insemination. Comparisons were made between fluid retention and biopsy score or age. In Experiment 2, a subset of 14 mares was classified for susceptibility to persistent breeding-induced endometritis in two subsequent breeding seasons. Biopsy score and age were associated with fluid retention (P < 0.001). In addition, age was related to biopsy score (P < 0.001). Of the mares examined for susceptibility, 36% (5 of 14) changed status during subsequent seasons. Three mares changed to a more severe classification (intermediate to susceptible, or resistant to intermediate), whereas two mares changed to a less severe classification (susceptible to intermediate).     

Night work,light exposure and melatonin on work days and days off

We aimed to examine the effects of night work on salivary melatonin concentration during and subsequent to night work and the mediating role of light. We included 254 day workers and 87 night workers who were followed during 322 work days and 301 days off work. Each day was defined as the 24 hour period starting from the beginning of a night shift or from waking in the mornings with day work and days off. Light levels were recorded and synchronized with diary information (start and end of sleep and work). On average, participants provided four saliva samples per day, and these were analyzed for melatonin concentration by liquid chromatography tandem mass spectrometry (LC-MS/MS). Differences between day and night workers on work days and days off were assessed with multilevel regression models with melatonin concentration as the primary outcome. All models were stratified or adjusted by time of day. For light exposure, we estimated the total, direct and indirect effects of night work on melatonin concentrations obtaining 95% confidence intervals through bootstrapping. On work days, night workers showed 15% lower salivary melatonin concentrations compared with day workers (?15.0%; 95% CI: ?31.4%; 5.2%). During the night, light exposure mediated a melatonin suppression of approximately 6% (?5.9%, 95% CI: ?10.2%; ?1.5%). No mediating effect of light was seen during the day time. On days off, we observed no difference in melatonin concentrations between day and night workers. These findings are in accordance with a transient and partly light-mediated effect of night work on melatonin production.     

Unusual Microbial Xylanases from Insect Guts

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted β-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.     

Low configurational stability of amfepramone and cathinone: Mechanism and kinetics of chiral inversion

The low configurational stability of two model compounds, the anorectic drug amfepramone and one of its basic metabolites, rac-cathinone, was examined for its mechanism and kinetics. Assuming a common intermediate for chiral inversion and deuteration, the rates of racemization were determined by the indirect method of proton–deuterium substitution monitored by ¹H-NMR. The rate of racemization increased linearily with increasing pD. At a pD of 7.4, the rate of racemization of both aminoketones was markedly dependent on buffer concentration, indicating a mechanism of general-base catalysis. The reactions were some five to six times faster in phosphate buffers than in hydroxylamine buffers of identical molarity. Amfepramone racemized about five times faster than the primary amine cathinone. One implication of these findings is that amfepramone and cathinone may be subject in vivo to chiral inversion catalyzed by numerous endogenous bases. As a result, it will be misleading to extrapolate rates of inversion expected in plasma from those measured in buffer solutions. © 1995 Wiley-Liss, Inc.     

Tissue kinetics,ion transport,and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm² (D_MRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO₃ (Ag) or methylene blue (MB). Part of the increased D_MRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (K_b) decreased and cell loss by moulting (K_d) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in D_MRC was accompanied by a 3-fold increase in Cl^- current (I_Cl). At day-2 there was a transient reduction in the I_Cl per MRC. H⁺ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased I_Cl and H⁺ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl^- (but not H⁺) transport capacity, was restored at day-7. We conclude that increased D_MRC following exposure to low external Cl^-, rather than being due to an increased K_b, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where morphological differentiation precedes functional differentiation.Parts of this study have been presented at the 32th International Congress of Physiological Sciences, 1–6 August, 1993, Glasgow, Scotland, and the 19th meeting of the European Study Group for Cell Proliferation, 5–9 October, 1993, Bruges, Belgium     

Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

Summary A cDNA library was constructed from poly(A)⁺RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)⁺-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.Abbreviations poly(A)⁺ polyadenylated - DS sodium dodecyl sulfate - D kilodalton - bp base pairs Supported by Research Grant GM 19807 from the United States Public Health Service and by additional funds from the University of California Research Council.     

Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit.

Biosynthesis of the phytohormone ethylene in higher plants proceeds via the following pathway: S-adenosylmethionine----1-aminocyclopropane-1-carboxylic acid (ACC)----ethylene. Ethylene-forming enzyme (EFE), the enzyme responsible for the oxidation of ACC to ethylene, has been only partially characterized in vitro. We have obtained authentic EFE activity in vitro from extracts of avocado fruit (Persea americana Mill. cv Hass). Ammonium sulfate fractionation revealed the presence of two EFE activities, which we designate as EFE1 and EFE2. EFE1 activity utilizes ACC and O2 as substrates and requires Fe(II) and ascorbate as cofactors. The enzyme has a relatively low Km (32 microM) for ACC, discriminates diastereomers of 1-amino-2-ethyl-cyclopropane-1-carboxylic acid, and is inhibited competitively by 2-aminoisobutyric acid, thus confirming its identity with authentic EFE. Activity is retained in a 100,000 x g supernatant and has a pH optimum of 7.5-8.0, suggesting a cytosolic localization.