alpha 1- and beta 2-adrenergic receptor expression in the Madin-Darby canine kidney epithelial cell line

The Madin-Darby canine kidney (MDCK) cell line, derived from distal tubule/collecting duct, expresses differentiated properties of renal tubule epithelium in culture. We studied the expression of adrenergic receptors in MDCK to examine the role of catecholamines in the regulation of renal function. Radioligand-binding studies demonstrated, on the basis of receptor affinities of subtype-selective adrenergic agonists and antagonists, that MDCK cells have both alpha 1- and beta 2- adrenergic receptors. To determine whether these receptor types were expressed by the same cell, we developed a number of clonal MDCK cell lines. The clonal lines had stable but unique morphologies reflecting heterogeneity in the parent cell line. Some clones expressed only beta 2-adrenergic receptors and were nonmotile, whereas others expressed both alpha 1- and beta 2-receptors and demonstrated motility on the culture substrate at low cell densities. In one clone, alpha- and beta- receptor expression was stable for more than 50 passages. Catecholamine agonists increased phosphatidylinositol turnover by activating alpha- adrenergic receptors and cellular cyclic adenosine monophosphate accumulation by activating beta-adrenergic receptors. Guanine nucleotide decreased the affinity of isoproterenol for the beta 2- receptor but did not alter the affinity of epinephrine for the alpha 1- receptor. These results show that alpha 1- and beta 2-receptors can be expressed by a single renal tubular cell and that the two receptors behave as distinct entities in terms of cellular response and receptor regulation. Heterogeneity of adrenergic receptor expression in MDCK clones may reflect properties of different types of renal tubule cells.     

N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.     

Zonal centrifugation and flotation– fractionation of MSD mutant mouse brain

Zonal centrifuge and flotation–fractionation profile analysis of neonatal mouse brain homogenates in iso-osmotic Ficoll–sucrose density–gradients demonstrates the presence of four light density fractions. In msd neurological mutant mice with a myelin-synthesizing deficiency syndrome, the bands appear to be relatively normal until after the 10th day of postnatal brain development. With the onset of visible neurological symptoms after the 11th day, the four density bands begin to disappear from the zonal profiles and are all but absent at the time of death at about the 21st postnatal day. In normal littermates of the mutants, the bands persist with age and intensify. Although their identities remain unknown, the top three identify by their density with adult myelin and the fourth with the lighter of two adult synaptosome fractions. Mixtures of brain homogenates between mutant and normal littermates give rise to zonal and banding profiles intermediate between the separate profiles but somewhat less than their average in intensity.     

Enantiospecific (S)-(+)-linalool formation from beta-myrcene by linalool dehydratase-isomerase

The linalool dehydratase-isomerase from Castellaniella defragrans strain 65Phen catalyzes in the thermodynamically unfavourable direction the hydration of betamyrcene to linalool and further the isomerization to geraniol, the initial steps in anaerobic beta-myrcene biodegradation. We have now investigated the stereochemistry of this reaction. (S)-(+)-Linalool is formed with an enantiomeric excess of at least 95.4%. (R)-(-)-Linalool was not detected. This indicates an introduction of the hydroxy group on the si-face of beta-myrcene.     

Slow Mitochondrial COI Sequence Evolution at the Base of the Metazoan Tree and Its Implications for DNA Barcoding

The evolution rates of mtDNA in early metazoans hold important implications for DNA barcoding. Here, we present a comprehensive analysis of intra- and interspecific COI variabilities in Porifera and Cnidaria (separately as Anthozoa, Hydrozoa, and Scyphozoa) using a data set of 619 sequences from 224 species. We found variation within and between species to be much lower in Porifera and Anthozoa compared to Medusozoa (Hydrozoa and Scyphozoa), which has divergences similar to typical metazoans. Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-evolving mtDNA is likely to be plesiomorphic for the Metazoa. Higher rates of evolution could have originated independently in Medusozoa and Bilateria or been acquired in the Cnidaria + Bilateria clade and lost in the Anthozoa. Low identification success and substantial overlap between intra- and interspecific COI distances render the Anthozoa unsuitable for DNA barcoding. Caution is also advised for Porifera and Hydrozoa because of relatively low identification success rates as even threshold divergence that maximizes the “barcoding gap” does not improve identification success. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function,Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.     

Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix: II. Further studies on the nature and site of transfilter “induction”

Corneal epithelial differentiation (primary stroma production) is dependent on the underlying extracellular matrix (ECM), for if the developing epithelium is enzymatically removed from the embryo, it fails to produce stroma in vitro unless it is cultured on collagenous ECM. We have previously shown that the stimulatory effect is mediated across Nucleopore filters in direct proportion to the surface area created by epithelial cell processes traversing the filter to contact ECM. Since collagenous ECM is insoluble under physiological conditions, transfilter stimulation of stroma production is probably due to an interaction of the epithelial cell surface with “inducer” ECM (killed lens capsule or purified collagen). We grew 5-day-old corneal epithelia on Nucleopore filters atop [³H]proline-labeled lens capsules and used both autoradiography and scintillation counting to show that radioactive collagen does not enter the epithelial cells in detectable amounts. We also show here that the stimulatory effect of collagen on collagen synthesis is not dependent on trapping of serum or binding of conditioned medium factors by ECM. Finally, we demonstrate that the stimulatory effect is reduced by removal of transfilter ECM after 6–12 hr in vitro. By 18–24 hr, however, cultured epithelium is less dependent on the substratum, probably because it has produced its own ECM. We conclude that: (1) the contact mediated collagen-cell surface interaction under study here requires the continuous presence of collagen in vivo and in vitro for maintenance of “stimulated” epithelial stroma synthesis; (2) the collagenous “inducer” interacts directly with epithelium rather than indirectly via trapped intermediates; (3) collagen acts at the epithelial cell surface without entering the cells.     

One-Way Allosteric Communication between the Two Disulfide Bonds in Tissue Factor

Tissue factor (TF) is a transmembrane glycoprotein that plays distinct roles in the initiation of extrinsic coagulation cascade and thrombosis. TF contains two disulfide bonds, one each in the N-terminal and C-terminal extracellular domains. The C-domain disulfide, Cys186-Cys209, has a ?RHStaple configuration in crystal structures, suggesting that this disulfide carries high pre-stress. The redox state of this disulfide has been proposed to regulate TF encryption/decryption. Ablating the N-domain Cys49-Cys57 disulfide bond was found to increase the redox potential of the Cys186-Cys209 bond, implying an allosteric communication between the domains. Using molecular dynamics simulations, we observed that the Cys186-Cys209 disulfide bond retained the ?RHStaple configuration, whereas the Cys49-Cys57 disulfide bond fluctuated widely. The Cys186-Cys209 bond featured the typical ?RHStaple disulfide properties, such as a longer S-S bond length, larger C-S-S angles, and higher bonded prestress, in comparison to the Cys49-Cys57 bond. Force distribution analysis was used to sense the subtle structural changes upon ablating the disulfide bonds, and allowed us to identify a one-way allosteric communication mechanism from the N-terminal to the C-terminal domain. We propose a force propagation pathway using a shortest-pathway algorithm, which we suggest is a useful method for searching allosteric signal transduction pathways in proteins. As a possible explanation for the pathway being one-way, we identified a pronounced lower degree of conformational fluctuation, or effectively higher stiffness, in the N-terminal domain. Thus, the changes of the rigid domain (N-terminal domain) can induce mechanical force propagation to the soft domain (C-terminal domain), but not vice versa.