Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model

The time dependency of the spontaneous aggregation of the fibrillogenic -Amyloid peptide, A^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-A antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 M amyloid peptide. The anti–A antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length A was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the A^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of A^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of A^1–40 in vitro and possibly in vivo.     

Steroid dehydrogenases in the adrenal gland of four species of birds: a histochemical study

Synopsis The presence of⁵-3-hydroxysteroid dehydrogenase, II-hydroxysteroid dehydrogenase, 17-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase has been demonstrated histochemically in the adrenal gland of the rain quailCoturnix coromendalica, barn owlTyto alba, brown crakeAmaurornis akool and painted partidgeFrancholinus pictus. All these enzymes occurred in the inter-renal cells. No activity was observed in the chromaffin cells. It is suggested that the inter-renal cells of these four species of birds are capable of synthesizing both corticosteroids and sex steroids.     

In-vivo phosphorylation of the cardiac L-type calcium channel beta-subunit in response to catecholamines

In canine myocardium, the -subunit of the L-type Ca²⁺ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217–222, 1993). We have assessed the identity of the -subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca²⁺ channel -subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the -subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dt_max. The results imply the involvement of a PKA-mediated phosphorylation of the Ca²⁺ channel -subunit in the adrenergic stimulation of intact canine myocardium.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

G-Protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca²⁺ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 M GTP-S did not significantly affect prepulse facilitation or whole-cell Ba²⁺ current (I _Ba) density. In contrast, inactivation of G proteins by intracellular GDP-S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of I _Ba, respectively. GDP-S dialysis resulted in nearly a threefold increase in peak I _Ba density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 m intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca²⁺ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (50%) in I _Ba amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca²⁺ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.This work was supported by a National Science Foundation grant (DCB-8812562), American Heart Association-Ohio Affiliate grant (SW-91-18), and an American Parkinson's Disease Association grant. C.A.D. was supported by a predoctoral National Research Service Award (National Institutes of Health training grant HL07571-08). The authors thank Kluener's Packing Co. for their generous supply of adrenal glands.     

Inhibition of R-factor-mediated β-lactamase by Proteus mirabilis

In cultures of Proteus mirabilis the level of R-factor-TEM-mediated -lactamase activity rose during the logarithmic phase, reached a maximum value at the end of this phase and fell rapidly when the cells entered the stationary phase. Supernatants from cultures in the stationary phase were found to inhibit R-TEM -lactamase activity both in cell-free preparations and in intact cells. The inhibitory activity may be due to a proteolytic enzyme excreted into the medium.     

The expression of I-Ed molecules in F1 hybrid mice detected with antigen-specific,I-Ed-restricted cloned T-cell lines

The expression of polymorphic determinants on I-E molecules is largely dependent on allelic variation in the E chain. We have previously analyzed the expression of E ^k and E ^b chains in F₁ hybrid mice by a combination of techniques, and have shown that functional variation detected by the responsiveness of cloned T-cell lines specific for these molecules correlates well with serological determination of E expression. In the present study, we have extended our analysis to E ^d expression in F₁ hybrid mice. We show that E ^d is relatively poorly expressed in three F₁ combinations: H-2 ^d× H-2 ^b, H-2 ^d× H-2 ^s, and H-2 ^d× H-2 ^u. The former two crosses express E chains from the H-2 ^dparent only; when recombinant strains carrying E ^b or E ^s and an active E gene are used, E ^d expression is significantly increased. On the other hand, H-2 ^umice synthesize E chains; the poor expression of E ^d chains in this F₁ hybrid apparently reflects the strong preferential association of E ^u chains with all E molecules thus far analyzed. These results confirm that E chains compete for binding to E chains and that preferential association of different allelic forms of E chains with E chains is a generalized phenomenon. They also illustrate the importance of the rate of biosynthesis of Ia chains for cell-surface expression.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

Microbial transformations in a cyclodextrin medium. Part 2. Reduction of androstenedione to testosterone by Saccharomyces cerevisiae

Summary An intensive and systematic investigation of the reductive transformation of androstenedione (AD) to testosterone by Saccharomyces cerevisiae was undertaken in the presence of natural and chemically modified cyclodextrins (CD). The bioconversion was significantly larger in the presence of - and -CD and hydroxypropyl--CD b but only slight in the presence of -CD and dimethyl- and trimethyl--CD. The performance of the various cyclodextrin media was interpreted in the light of the measured phase solubility diagrams of AD. Further investigation focused on biotransformation of the -CD-androstenedione complex, the formation of which was studied by differential scanning calorimetry and X-ray powder diffractometry and stoichiometry determined by ¹H-nuclear magnetic resonance. A mechanism whereby CDs reduce the effective inhibitory concentrations of substrate and product as well as facilitate transport of the complexed substrate through the yeast cell wall has been suggested for the CD-promoted biotransformation. Offprint requests to: R. Bar     

Assembly-dependent expression of antigenic epitopes by β2-microglobulinb

In this report we provide evidence for the expression of antigenic epitopes on mouse (₂-microglobulin^b _2m ^b) that result from assembly with cognate H-2 class I heavy chains. For the cell line 69.9.15 (₂m^a × ₂m^b), which expresses a mutant cytosolic form of H-2K^b and wild-type H-2D^b, flow cytometry with rabbit antiserum against mouse ₂m displayed ₂m expression by cells grown in the presence or absence of fetal calf serum. By contrast, the epitopes identified by the ₂m^b-specific monoclonal antibody (mAb) S19.8 and clone 23 were not expressed by 69.9.15 cells grown in serum-containing conditions, and although S19.8 reactivity was weakly recovered by culture in the absence of serum, no such reacitivity was observed with clone 23. Strong expression of these epitopes was achieved following transfection of 69.9.15 cells with the wild-type H-2K ^b gene, indicating that the ₂m^b epitopes defined by mAb S19.8 and clone 23 were expressed when ₂m^b was assembled with an appropriate heavy chain. In support of this conclusion, we observed the recovery of the S19.8 and clone 23 epitopes by in vitro assembly of H-2K^b heavy chains with ₂m^b in the presence of the VSV N protein p52–59; however, such epitopes were expressed neither by ₂m^b prior to heterodimer assembly nor by non-conformed ₂m^b present in tissue culture supernatants recovered from H-2 class I surface positive cells. Taken together, these data indicate that in addition to the property of ₂m to modify the antigenicity of the MHC class I heavy chains, ₂m epitopes are induced in a reciprocal manner by assembly with MHC class I heavy chain molecules. Correspondence to: R. A. Zeff.     

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I -1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5-deletion analysis of the class I -1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I -1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I -1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I -1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these data strongly suggest that ethylene is signalling ozone-induced expression of class I -1,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an additional ethylene independent signalling pathway for ozone-induced expression of genes involved in phytoalexin biosynthesis.     

Stereo-isomer specific induction of renal cell apoptosis by synthetic muramyl dipeptide lpar;N-acetylmuramyl-L-alanyl-D-isoglutamine)

In order to understand the modification of -adrenoceptor linked signal transduction by changes in the intracellular Ca²⁺, we examined the status of -adrenoceptors (-ARs), G-proteins and adenylyl cyclase (AC) in Ca²⁺-deficiency and Ca²⁺-overload by perfusing the isolated rat heart with Ca²⁺-free medium for 5 min and Ca²⁺-containing medium for 5 min following Ca²⁺-free perfusion, respectively. Ca²⁺-depletion caused not only an increase in basal, isoproterenol-, Gpp(NH)p-, NaF- and forskolin-stimulated AC activities but also produced an increase in the ₁-AR affinity and density as well as up-regulation of G_s-protein function and uncoupling of G_i-protein to AC. Ca²⁺-repletion for 5 min following 5 min Ca²⁺-free perfusion reversed the increased AC activities to varying degrees. The ₁-AR affinity was further increased upon Ca²⁺-repletion whereas its density was decreased. Ca²⁺-repletion also decreased protein content for AC and -AR kinase but augmented the changes in G_s- and G_i-protein functions. Although low Na⁺- medium perfusion during Ca²⁺-depletion prevented the changes in G-proteins during both Ca²⁺-depletion and Ca²⁺-repletion periods, the increased ₁-AR affinity and density as well as changes in AC activities due to Ca²⁺-depletion were not affected while alterations due to Ca²⁺-repletion were fully prevented. These results suggest that changes in Ca²⁺-homeostasis may represent a mechanism for alterations in the -adrenergic signal transduction pathway in the heart under pathological conditions.     

Hormonal regulation of hepatic glycogenolysis inAmphibolurus nuchalis,the western netted dragon: an in vitro study

Summary In mammals hepatic glycogenolysis is controlled by several hormones using cyclicAMP, Ca²⁺ and/or diacylglycerol as intracellular messengers. In contrast, in teleost fish, lungfish and amphibians fewer hormones promote hepatic glycogenolysis, and cyclicAMP is the sole intra-cellular messenger. This suggests that the -adrenergic mechanism became associated with the liver after amphibians separated from the vertebrate line. Reptiles separated later, and the aim of this study is to elucidate the hormonal control of hepatic glycogenolysis in a reptile,Amphibolurus nuchalis, and especially to determine which adrenergic receptor system is operative.InA. nuchalis liver pieces cultured in vitro, adrenaline and glucagon stimulated glycogen breakdown and glucose release, glycogen phosphorylase activity and accumulation of cyclicAMP in the tissue. Neurohypophysial peptides did not affect these parameters. These actions of adrenaline were completely blocked by the -adrenergic antagonist, propranolol and slightly reduced by the -adrenergic antagonist, phentolamine. Removal of Ca²⁺ from the medium and addition of the Ca²⁺ chelator, EGTA, did not block the actions of adrenaline, and the Ca²⁺ ionophore A23187 did not mimic these actions.The -adrenegic ligand [¹²⁵I]-iodocyanopindolol (ICP) bound specifically to an isolated membrane preparation fromA. nuchalis liver with a calculated K_D of 100 pM and a Bmax of 37.6 fmol·mg protein^–1. The adrenergic ligands propranolol, isoprenaline, adrenaline, noradrenaline, phenylephrine and phentolamine displaced ICP with K_D's of 20 nM, 1 M, 4.5 M, 32 M, 35 M and 500 M, respectively. The ₂-adrenergic ligand yohimbine did not bind specifically to the membrane, but at 1 nM and 100 pM, specific binding of the ₁-adrenergic ligand prazosin was 45% of total with a mean of 11.3 fmoles·mg protein^–1 specifically bound.These findings indicate that the glycogenolytic actions of adrenaline are mediated primarily via -adrenergic receptors inA. nuchalis, but that -adrenergic receptors may also play some role in the control of hepatic metabolism.     

Lack of specificity in cation effects on solubilized benzodiazepine receptor

Receptors for benzodiazepines (BZ) and -carboline-carboxylic acid ethyl ester (-CCE) has been solubilized with decanoly-N-methylglucamide (DMG), a new kind of nonionic detergent. The apparent dissociation constants of diazepam and -CCE for solubilized receptor were similar to those for synaptic membranes. Sucrose density gradient centrifugation of the solubilized receptor protein revealed that the binding profile of [³H]-CCE essentially parallels that of [³H]diazepam and that both sedimentation coefficients were 10.5S. Co²⁺ and Ni²⁺, which increase [³H]diazepam binding and decrease [³H]-CCE binding to synaptic membranes, remarkably increased the binding of both to the solubilized receptor. Mg²⁺ and Ca²⁺, which had no effect on membrane receptor binding, also enhanced [³H]diazepam and [³H]-CCE binding to the solubilized receptor. The increase in binding in the presence of these divalent cations was due to a change in the apparent number of binding sites, with no change in binding affinities. The relative lack of specificity in divalent cation effects on solubilized BZ receptor may be caused by separation or destruction of the cation recognition site or channel of the BZ receptor complex by solubilization of the synaptic membrane with DMG.     

Secondary structure determination by NMR spectroscopy of an immunoglobulin-like domain from the giant muscle protein titin

Summary We present the complete ¹⁵N and ¹H NMR assignment and the secondary structure of an immunoglobulin-like domain from the giant muscle protein titin. The assignment was obtained using homonuclear and ¹⁵N heteronuclear 2D and 3D experiments. The complementarity of 3D TOCSY-NOESY and 3D ¹⁵N NOESY-HSQC experiments, using WATERGATE for water suppression, allowed an efficient assignment of otherwise ambiguous cross peaks and was helpful in overcoming poor TOCSY transfer for some amino acids. The secondary structure is derived from specific NOEs between backbone - and amide protons, secondary chemical shifts of -protons and chemical exchange for the backbone amide protons. It consists of eight -strands, forming two -sheets with four strands each, similar to the classical -sandwich of the immunoglobulin superfamily, as previously predicted by sequence analysis. Two of the -strands are connected by type II -turns; the first -strand forms a -bulge. The whole topology is very similar to the only intracellular immunoglobulin-like domain for which a structure has been determined so far, i.e., telokin.     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, J., Hanisch, F.-G., Neumann, U. (1991)Glycoconjugate J 8:414–23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a new ganglioside, supposed to represent GalNAc-G_D1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).