Mechanisms of fluorophore formation in the histochemical glyoxylic acid method for monoamines

Summary Glyoxylic acid vapour is a most powerful reagent for the fluorescence histochemical visualization of biogenic monoamines. In the present investigation the mechanisms of fluorophore formation in the glyoxylic acid reaction has been studied in detail for tryptamine in histochemical models and in freeze-dried tissue, utilizing microspectrofluorometric, Chromatographic, and mass spectrometric techniques in combination with isotope measurements.The glyoxylic acid-tryptamine reaction proceeds through an initial Pictet-Spengler type cyclization to 1,2,3,4-tetrahydro--carboline-1-carboxylic acid, followed by two alternative fluorophore forming reactions yielding 3,4-dihydro--carboline, or the 2-carboxymethyl-3,4-dihydro--carbolinium and 2-methyl-3,4-dihydro--carbolinium salts, which are all strongly fluorescent. It is shown that the yield of fluorophores is considerably higher in the glyoxylic acid vapour reaction than in the formaldehyde vapour reaction of the standard Falck-Hillarp method, and that this higher efficiency of glyoxylic acid is due to the most favourable catalysing properties of the carboxylic group of the glyoxylic acid molecule.     

Intrinsic fluorescence studies on saccharide binding toArtocarpus integrifolia lectin

The combining region ofArtocarpus integrifolia lectin has been studied by using the ligand-induced changes in the fluorescence of the lectin. The saccharide binding properties of the lectin show that C-l, C-2, C-4, and C-6 hydroxyl groups of D-galactose are important loci for sugar binding. The -anorner of galactose binds more strongly than its -counterpart. Inversion in the configuration at C-4 as in glucose results in a loss of binding to the lectin. The C-6 hydroxyl group is also presumably involved in binding as D-fucose does not bind to the lectin.The lectin binds to the Thomsen-Friedenreich antigen (Gal(13)GalNAc) more strongly than the other disaccharides studied, viz. Gal/ (14) Gal and Gal (13) GlcNAc, which are topographically similar to T-antigen. This observation suggests that the combining region ofArtocarpus lectin is complementary to that of T-antigen.Solvent accessibility of the protein fluorophores have been probed by the quenching of protein fluorescence by Iodide ion in the absence and presence of sugar. In the presence of sugar a slight inaccessibility of the fluorophores to the solvent has been observed.Abbreviations MeGal 1-O-methyl--glucopyranoside - MeGal 1-O-methyl--glucopyranoside - GalNAc 2-acetamido-2-deoxy-galactose - Gal Galactose     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

A critical evaluation of the specificity of aniline blue induced fluorescence

Summary Commercial aniline blue dyes are heterogeneous and variable. We have isolated and purified the fluorochrome from water soluble aniline blue. This fluorochrome fluoresces weakly with a maximum emission around 455 nm but the fluorescence is shifted to longer wavelengths (500–506 nm) when complexed with isolated -1,3-glucans, cellulose or mixed-linked glucans. A similar intense fluorescence is observed in sieve plates, new cell walls and around pitfields in the presence of the fluorochrome. The fluorescence induced by the aniline blue fluorochrome does not specifically indicate the presence of -1,3-glucans. Indeed most wall features are induced to fluoresce to some extent by the fluorochrome. However, fluorescence is modified by lignins and phenolics. Furthermore the intense fluorescence induced in the traditional callose sites, sieve plates, around pitfields and in new cell walls is probably related to localized differences in the structural packing of wall polymers.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Media from Rhabdomyosarcoma and Neuroblastoma Cell Cultures Stimulate in Vitro Aggregation and Fibrillization of Amyloid Beta-Protein

In vitro aggregation and fibrillization of synthetic amyloid beta-protein A 1–40 was assessed in the conditioned media from rhabdomyosarcoma (CRL 1598, HTB 82, HTB 153, CCL 136), adenocarcinoma (CCL 218), neuroblastoma (SY5Y), and COS cells cultured in the absence and presence of 10% heat-inactivated fetal bovine serum (FBS). The aggregation and formation of cross -pleated sheet structures in A was quantitated by Thioflavin T (ThT) fluorescence spectroscopy, while the morphology of A fibrils was examined in negative staining in the electronmicroscope (EM). In cultures supplemented with 10% FBS, the conditioned media from CRL 1598, HTB 82, CCL 218, and SY5Y cell cultures stimulated A aggregation in a time-dependent manner as compared to that of control (serum-containing medium that had not been exposed to cells). The order of stimulation was SY5Y > CRL 1598 HTB 82 > CCL 218, and the stimulation was higher in 2 week cultures than in 1 week cultures. Similar studies using media from HTB 153, CCL 136 and COS cell cultures showed no effect on A 1–40 aggregation. In serum-free cell cultures, only media from SY5Y and CRL 1598 could promote significant aggregation of A 1–40. Negative staining in EM revealed A fibril formation only with conditioned media from SY5Y and CRL 1598 cultured under serum free conditions; no A fibrils were noticed in media from cell cultures supplemented with 10% FBS. We propose that both the SY5Y neuroblastoma cell line and the CRL 1598 rhabdomyosarcoma cell line may serve as experimental models for in vitro studies of extracellular aggregation and fibrillization of A-protein in cell cultures, while rhabdomyosarcoma HTB 82 and adenocarcinoma CCL 218 may be models for study of A aggregation only.     

Immunohistological analyses of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, G_M3(Neu5Ac), G_M3(Neu5Gc) and G_M1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was G_M3(Neu5Ac) followed by moderately expressed G_M3(Neu5Gc) and G_M1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-G_M3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle. Abbreviations: BSA, bovine serum albumin; DAPI, 4, 6-diamidine-2-phenylindole-dihydrochloride; DTAF, dichlorotriazinylamino-fluorescein; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycolylneuraminic acid [53]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [54] and the nomenclature of Svennerholm [55]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gal1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₃Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₅Cer, GalNAc1-3GalNAc1-3GAl1-4Gal1-4Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer.     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.     

A gene encoding Achlya bisexualis β-amylase and its expression in Saccharomyces cerevisiae

Part of a -amylase genomic DNA sequence from the oomycete, Achlya bisexualis was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotide primers derived from the conserved regions of other known -amylase sequences. The 5- and 3-regions of the -amylase gene were amplified by genome walking method. The Ach. bisexualis -amylase gene consisted of a 1338bp open reading frame, encoding a protein of 446 amino acids with a molecular weight of 49 381Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 67% similarity to the -amylase of Saprolegnia ferax, followed by 40% similarity to that ofArabidopsis thaliana. The -amylase gene was expressed in Saccharomyces cerevisiae placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.     

Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein -casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas12) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human -casein cDNA. The presence of human -casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human -casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human - casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human -casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as -casein. The above experiments demonstrate the expression of human milk - casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children     

The recognition of three different epitopes for the H-type 2 human blood group determinant by lections ofUlex europaeus,Galactia tenuiflora andPsophocarpus tetragonolobus (Winged Bean)

The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc(1–2)Gal(1–4)GlcNAcMe) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc(1–2)Gal(1–4)XylMe was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.Abbreviations Me methy - Bn benzyl - Ac acetyl - Bz benzoyl - n-Bu n-butyl - NMR nuclear magnetic resonance - the GlcNAc Gal and Fuc residues of the H-type 2 trisaccharide are designated as the a, b and c structural units, respectively. This is paper XV in a series devoted to molecular recognition.     

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     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

Decreases in Plasma Aβ1-40 Levels with Aging in Non-Demented Elderly with ApoE-ε4 Allele

This report examines plasma amyloid proteins A40 and A42 and apolipoprotein E (apoE) levels and their relationships with age in non-demented older adults with (N = 32) or without the apoE-4 allele (N = 94). A levels did not differ between the groups whereas the 4 allele was associated with a significant reduction in plasma apoE. In subjects with the 4 allele, increasing age was associated with significant reduction in plasma A40. Subjects without the 4 allele showed a significant positive correlation between A40 and A42 levels. There was also a significant correlation between plasma A40 and apoE levels in all subjects.     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)