Leukotriene-related gene polymorphisms in ASA-intolerant asthma: an association with a haplotype of 5-lipoxygenase

A recent study has demonstrated the possible involvement of a leukotriene C4 synthase (LTC4S) gene polymorphism in ASA-intolerant asthma (AIA) in a Polish population, whereas no significant association was noted in other populations. To investigate the role of genetic polymorphism in AIA development, we screened single nucleotide polymorphisms (SNPs) of the key enzymes involved in arachidonate metabolism, and the cysteinyl leukotriene receptor 1 (CYSLTR1) in a large Korean population with AIA: 93 AIA and 181 ASA-tolerant asthma (ATA) patients, and 123 normal controls. The single-base extension method was used to genotype SNPs in 5-lipoxygenase (ALOX5, –1708GA, 21CT, 270GA, 1728GA), ALOX5-activating protein (ALOX5AP, 218AG), prostaglandin-endoperoxide synthase 2 (PTGS2, COX2, –162CG, 10TG, R228H, V511A), LTC4S (–444AC), and CYSLTR1 (927TC). Haplotype analyses were undertaken for the SNPs in ALOX5. No significant differences in allele and genotype frequencies of single SNPs were observed between the patient groups (P>0.05). However, the frequency of the ALOX5-ht1[G-C-G-A] haplotype in the AIA group was significantly higher than its frequency in the ATA group with a probability (P) of 0.01, odds ratio (OR) of 5.0, and 95% confidence interval (95%CI) of 1.54–17.9, and in the normal controls (P=0.03, OR=4.5, 95%CI=1.1–18.4), by using a dominant model. These results suggest a lack of association between the ALOX5AP, PTGS2, LTC4S, and CYSLTR1 gene polymorphisms and the AIA phenotype in the Korean population. However, the possible involvement of ALOX5-ht1[G-C-G-A] in AIA development is suggested.J.-H. Choi and H.-S. Park contributed equally to this work as first authors     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal,normal and neoplastic gastrointestinal tract

Summary Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for 26 sialylated type 2 chain) and IB9 (for the 26 sialylated type 2 chain and glycoproteins having NeuAc26GalNAc), and 188C1 (for short- and long-chain Le^x antigens) and FH2 (for the long-chain Le^x antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The 26 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Le^x antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its 26 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Le^x antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc26GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.Abbreviations Fuc fucose - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - MAb monoclonal antibody - NeuAc N-acetylneuraminic acid - PBS phosphate-buffered saline     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Seven members of the (1→3)-β-glucanase gene family in barley (Hordeum vulgare) are clustered on the long arm of chromosome 3 (3HL)

Members of the (13)--glucan glucanohydrolase (EC 3.2.1.39) gene family have been mapped on the barley genome using three doubled haploid populations and seven wheat-barley addition lines. Specific probes or polymerase chain reaction (PCR) primers were generated for the seven barley (13)--glucanase genes for which cDNA or genomic clones are currently available. The seven genes are all located on the long arm of chromosome 3 (3HL), and genes encoding isoenzymes GI, GII, GIII, GIV, GV and GVII (ABG2) are clustered in a region less than 20 cM in length. The region is flanked by the RFLP marker MWG2099 on the proximal side and the Barley Yellow Mosaic Virus (BYMV) resistance gene ym4 at the distal end. The gene encoding isoenzyme GVI lies approximately 50 cM outside this cluster, towards the centromere. With the exception of the gene encoding isoenzyme GIV, all of the (13)--glucanase genes are represented by single copies on the barley genome. The probe for the isoenzyme GIV gene hybridized with four DNA bands during Southern blot analysis, only one of which could be incorporated into the consensus linkage map.     

Biosynthesis of brassinolide from teasterone via typhasterol and castasterone in cultured cells of Catharanthus roseus

The biosynthesis of brassinolide (BL) in crown gall and nontransformed cells of Catharanthus roseus in which BL, castasterone (CS), typhasterol (TY), and teasterone (TE) are endogenous was investigated using deuterated TY and TE as substrates. The metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and/or GC-selected ion monitoring (SIM). It was found that these cells converted TY to CS and BL, as well as TE to TY and CS. Because the pathway from CS to BL in the cells has already been confirmed, a biosynthetic sequence of TE TY CS BL was established. Reversible conversion between TE and TY was observed.Biosynthesis of brassinosteroids in Catharanthus roseus. Part III. Part II of this series: Suzuki et al. (1993).     

Haplotype analysis of familial amyloidotic polyneuropathy

Summary Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP anyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution ³⁰ValMet has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the ValMet mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the ValMet mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the ValMet mutation can be explained by a C-T transition at the C_pG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the ValMet mutation has probably recurred in the human population, to generate FAP families of independent origin.     

Assessing glycosidic linkage flexibility: Conformational analysis of the repeating trisaccharide unit of Aeromonas salmonicida

Summary A detailed conformational analysis was performed for the synthetic branched trisaccharide -d-Man-NAc-(14)-[-d-Glc-(13)]-l-Rha 1 which represents the repeating unit of the O-antigenic polysaccharide of Aeromonas salmonicida. The study was based on 26 experimental NOE curves from 1D transient NOE experiments, employing Gaussian-shaped inversion pulses at 600 MHz. Eight of the NOE curves were interglycosidic and thus useful for an analysis of glycosidic linkage orientations. Metropolis Monte Carlo (MMC) simulations and minimum-energy calculations with the program GEGOP were used to obtain theoretical NOE curves which were compared to the experimental ones. MMC simulations with different temperature parameters of 310, 600, 900 and 2000 K allowed identification of NOEs which are sensitive towards different conformation distributions-not only different conformations-at both glycosidic linkages in 1. A comparison of trisaccharide 1 with the constituent disaccharides -d-ManNAc-(14)-l-Rha 2 and -d-Glc-(13)-l-Rha 3 revealed effects of branching on glycosidic linkage flexibility. A quantitative evaluation was facilitated by the introduction of entropy-related flexibility parameters. Our study indicates a notable restriction of flexibility, especially at the (13) linkage in 1. Although overall flexibility in 1 is reduced as compared to the constituent disaccharides 2 and 3, it cannot be neglected altogether. In summary, combined transient NOE experiments and MMC simulations provide a simple approach to analyse glycosidic linkage flexibility.     

Localization of hydrogenase and nitrate reductase in Campylobacter sputorum subsp. bubulus

Campylobacter sputorum subsp. bubulus contained hydrogenase activity after growth with lactate and nitrate and after growth with hydrogen and nitrate. After growth with hydrogen and nitrate a molar growth yield (g dry cells/mol hydrogen) of 5.6 was measured. Hydrogenase and nitrate reductase were membrane-bound enzymes. In cells with high hydrogenase activity the H⁺/O, H⁺/NO _inf2 ^sup- and H⁺/NO _inf3 ^sup- values with hydrogen as the electron donor were 3.74, 2.61 and 4.36 respectively. In cells with low hydrogenase activity these values were 2.33,-0.86 and 1.31 respectively. These values and the stoichiometry of respiration-driven proton translocation (H⁺/2e=2) led to the conclusion that hydrogenase is located at the periplasmic side of the cytoplasmic membrane. In cells with low lactate dehydrogenase activity or low hydrogenase activity the reduction of nitrate to nitrite could be separated from the reduction of nitrite to ammonia. Positive H⁺/NO _inf3 ^sup- values (between 0.9 and 1.7) with lactate or hydrogen as the electron donor were measured in these cells whereas H⁺/NO _inf2 ^sup- values were negative. From this result it was concluded that nitrate reductase is located at the cytoplasmic face of the cytoplasmic membrane. The results explain the previous observation that molar growth yields with nitrate were somewhat higher than those with nitrite.     

Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus

Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H⁺/O values and H⁺/NO ₂ ^- values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H⁺/2e value of 2.0. The H⁺/O and H⁺/NO ₂ ^- values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MTPP⁺ methyltriphenylphosphonium cation - TMPD N,N,N,N-tetramethyl-p-phenylenediamine; H⁺/O (H⁺/NO ₂ ^- ), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO ₂ ^- ); H⁺/2e (q⁺/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor     

Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Metabolism ofL-sorbose by enzymes fromGluconobacter melanogenus IFO 3293

Summary Studies on the metabolism ofL-sorbose by cell-free extracts fromGluconobacter melanogenus IFO 3293 grown inL-sorbose — containing media confirm by different methodology the observations by Makover et al. that the sequence in this bacterium isL-sorbose L-sorbosone 2-keto-L-gulonic acid L-idonic acid.Dedicated to the memory of Prof. Dr. Konrad Bernhauer. For preceeding paper in this series see Kitamura & Perlman (1975).     

Interstitial deletion of the short arm of chromosome 3

Summary A de novo interstitial deletion of the short arm of chromosome 3 was prenatally diagnosed in a male fetus, karyotype 46,XY,del(3)(pterp14.2::p11qter). The fetus had craniofacial dysmorphisms, a single transverse palmar crease, ulnar deviation in the wrists, cardiovascular anomalies, a slight ureteric dilatation and a mobile caecum. Our observations are compared with five other cases with interstitial deletion of the short arm of chromosome 3 to delineate further the proximal 3p deletion syndrome. The gene for beta-galactosidase-1 (GLB-1) has previously been assigned to chromosome 3(p21q21). The absence of a gene dosis effect for GLB-1 in this study indicates exclusion of GLB-1 from 3(p11p14.2).     

Structure-function relationships of β- D-glucan endo- and exohydrolases from higher plants

(13),(14)--d-Glucans represent an important component of cell walls in the Poaceae family of higher plants. A number of glycoside endo- and exohydrolases is required for the depolymerization of (13),(14)--d-glucans in germinated grain or for the partial hydrolysis of the polysaccharide in elongating vegetative tissues. The enzymes include (13),(14)--d-glucan endohydrolases (EC 3.2.1.73), which are classified as family 17 glycoside hydrolases, (14)--d-glucan glucohydrolases (family 1) and -d-glucan exohydrolases (family 3). Kinetic analyses of hydrolytic reactions enable the definition of action patterns, the thermodynamics of substrate binding, and the construction of subsite maps. Mechanism-based inhibitors and substrate analogues have been used to study the spatial orientation of the substrate in the active sites of the enzymes, at the atomic level. The inhibitors and substrate analogues also allow us to define the catalytic mechanisms of the enzymes and to identify catalytic amino acid residues. Three-dimensional structures of (13),(14)--d-glucan endohydrolases, (14)--d-glucan glucohydrolases and -d-glucan exohydrolases are available or can be reliably modelled from the crystal structures of related enzymes. Substrate analogues have been diffused into crystals for solving of the three-dimensional structures of enzyme-substrate complexes. This information provides valuable insights into potential biological roles of the enzymes in the degradation of the barley (13),(14)--d-glucans during endosperm mobilization and in cell elongation.     

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

Center for barr body condensation. A case of Turner's syndrome with 45,X/46,X,dic(X) (Xqter→ p22::p22→qter)

Summary An 11-year-old girl with karyotype 45,X/46,X,dic(X) (Xqterp22::p22qter) is presented. The abnormal X is always found to be the inactive and late replicating X, and according to previous investigations by Therman et al. (1974) part of the cells are seen to have bipartite Barr bodies.     

A Comparison of the Lipopolysaccharides and O-Specific Polysaccharides of Azospirillum brasilense Sp245 and Its Omegon-Km Mutants KM018 and KM252

The lipopolysaccharides (LPSs) extracted from the outer membrane of Azospirillum brasilense Sp245 and its Omegon-Km mutants KM018 and KM252 with a hot aqueous solution of phenol were found to differ in the content of carbohydrates, glucosamine, and total phosphorus and in the proportion of octadecenoic and hexadecanoic acids in the lipid moieties of the LPSs. The carbohydrate moieties of the LPSs were heterogeneous in charge. The analysis of the O-specific polysaccharides (O-PSs) of the mutants KM018 and KM252 by gas–liquid chromatography, IR spectroscopy, and NMR spectroscopy showed that they are composed of the same linear pentasugar repeating units 2)--D-Rhap-(1 3)--D-Rhap-(1 3)--D-Rhap-(1 2)--D-Rhap-(1 2)--D-Rhap-(1 as the O-PSs of the parent strain Sp245. The reported differences in the biological activity of the LPSs of the parent and mutant strains can be due to their different chemical composition.     

Regional localization of the human genes for S-adenosylhomocysteine hydrolase (cen→q131) and adenosine deaminase (q131→qter) on chromosome 20

Summary The gene loci for S-adenosylhomocysteine hydrolase (AHCY) and adenosine deaminase (ADA), two enzymes with related metabolic functions, have both been assigned to human chromosome 20. We have used rodent-human somatic hybrids containing translocations involving human chromosome 20 to more precisely determine the relative locations of the AHCY and ADA loci. Our results assign the AHCY locus to the long arm of chromosome 20, in the region cenq131, and ADA to the region q131qter.     

High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations

Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96 Asn; PM 374: Asp96 Gly; PM 384: Asp85 Glu) were grown in a synthetic medium containing (4-¹³C)-Asp. The mutant bacteriorhodopsins labeled with (4-¹³C)-Asp (37%–45%), and owing to the metabolism of Halobacteria also with (11-¹³C)-Trp (50%–100%), were isolated as purple membranes and ¹³C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989 a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85 Glu) has an absorption maximum at 610 run. It can be converted into a purple form (_max = 5.40 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed.Abbreviations CHAPSO 3-(3-cholamidopropyl)-dimethylammonio2-hydroxy-1-propane sulfonate - CP crosspolarization - bR bacteriorhodopsin - FTIR Fourier-transform-infrared - FID free induction decay - IR Infrared - MASS Magic angle sample spinning - NMR Nuclear magnetic resonance - TMS tetramethylsilane This research was supported by the Deutsche Forschungsgemeinschaft #SFB 60G9 Offprint requests to: M. Engelhard