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PubMed1994

The human EPRS locus (formerly the QARS locus): a gene encoding a class I and a class II aminoacyl-tRNA synthetase.

Glutamyl-tRNA synthetase and prolyl-tRNA synthetase belong to different classes of aminoacyl-tRNA synthetases that are thought to have evolved along independent evolutionary pathways. However, both enzymes are on one polypeptide chain encoded by a single human gene, the EPRS locus, which is transcribed as one long mRNA. We report the structure of the human EPRS gene, which consists of 29 exons spread over at least 90 kb of genomic DNA. The exons, encoding the glutamyl-specific and the prolyl-specific parts of the enzyme, are each clustered in 10-kb sections located at opposite ends of the gene. These two exon clusters are separated by a long intervening DNA section with a number of exons, encoding functions that may be involved in the organization of the mammalian multienzyme synthetase complex. The upstream gene region shows structural features of a regulated gene, and preliminary experiments suggest that the gene is expressed at specific times in growth-stimulated cultured cells. We have localized the gene to the distal long arm of human chromosome 1 and to a corresponding site in mouse chromosome 1.

Amino Acid Sequence↗
PubMed2005

Military fitness class of Finnish 18-year-old men--prediction of military fitness class at call-up with the YASR and sociodemographic factors.

OBJECTIVE: The aim of this study was to examine psychosocial factors associated with military fitness class, classified as capability of service, vs. temporary or permanent exemption from service. METHOD: Participants were 2,340 (80% of the original sample) Finnish 18-year-old men at call-up for obligatory military service who filled in the Young Adult Self-Report (YASR) questionnaire. RESULTS: Temporary exemption from military service was independently associated with the YASR total problem score, externalizing and internalizing problems, excessive alcohol use, drug use, problems with peers and family, and mental health service use. Permanent exemption was independently associated with problems with peers and family. CONCLUSION: The YASR is a potential selection tool to be used at call-up. Temporary exemption was more strongly associated with psychosocial problems than permanent exemption. Because of the high level of psychological problems among those temporarily exempted, the call-up situation offers a unique opportunity to identify those with problem behaviour and risk of marginalization.

Adolescent↗
PubMed2006

WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis.

The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37 degrees C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Delta mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell-daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2 Delta strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25 degrees C and was not viable at 37 degrees C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.

Animals↗
PubMed1982

Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

Animals↗
PubMed1991

Detection of two different anti-factor VIII/von Willebrand factor antibodies of the IgA class in a hemophilic patient with a polyclonal factor VIII inhibitor of the IgG class.

A hemophilic patient treated with Factor VIII (F.VIII) concentrates developed a F.VIII inhibitory activity. The patient's plasma showed 2 anti-F.VIII/von Willebrand Factor (vWF) antibodies (Abs) of the IgG and IgA class respectively. The specific anti-F.VIII/vWF Abs were isolated and resulted to be IgG (20 micrograms/ml) and IgA (35 micrograms/ml). The plasma was subsequently fractionated by Protein A-Sepharose and the IgG and IgA containing fractions were separately analyzed for anti-F.VIII activity. Both fractions exhibited F.VIII inhibitory activity, but that corresponding to the IgA was lower than expected. Regarding the possible existence of a non-inhibitor Ab in the IgA containing sample, plasma was processed through an immunoadsorbent to which a F.VIII devoid vWF preparation had been previously coupled and further fractionated by Protein A-Sepharose, obtaining 2 IgA fractions. One of them exhibited F.VIII inhibitory activity while the other, which reacted with the F.VIII devoid vWF preparation, did not. Therefore, this latter one was considered as a true anti-vWF Ab. The IgG and IgA inhibitors were polyclonal and the IgG one was composed of the 4 IgG subclasses.

Adult↗
PubMed1990

Both HLA class II and class III DNA polymorphisms are linked to juvenile rheumatoid arthritis susceptibility.

HLA-DR5 and DRw8 are not significantly linked to juvenile rheumatoid arthritis (JRA) in the Spanish population. However, we have found restriction fragment length polymorphisms (RFLPs) beta DR-2.9-kb-EcoRI and C4-14.3-kb-EcoRI, that correlate significantly with JRA. Eighty-six percent of our patients present either HLA-DR5 or the beta DR-2.9-kb-EcoRI allogenotope or lack the C4-14.3-kb-EcoRI allogenotope compared to 48% in controls; however, these particular DR and C4 RFLPs may not represent the corresponding DRB1 or C4 genes but rather neighboring ones which may be relevant to confer JRA susceptibility. The C4-14.3-kb-EcoRI RFLP decrease in patients may reflect a decrease in C4 B "long" phenotypes. Also, both RFLPs are the only markers able to identify most of the JRA patients in our population. On the other hand, the findings of both class II and III genes associated to JRA may reflect the existence of epistatic interactions (cis or trans) within the HLA gene cluster which lead to a particular pathology in certain diseases.

Arthritis, Juvenile↗
PubMed1986

An improved biochemical method for the analysis of HLA-class I antigens. Definition of new HLA-class I subtypes.

A simple method is described for the biochemical analysis of HLA class I antigens. It is a modification of a previously published procedure for one-dimensional isoelectric focusing (1D-IEF), giving improved resolution and offering larger sample capacity. One million viable cells suffice for analysis, and no more than 25 muCi of radioisotope (35S-methionine) are required. The usefulness of the method is illustrated by the characterization of a total of four biochemically distinct subtypes of HLA-B27, three subtypes of HLA-A24, two subtypes of HLA-A11, three subtypes of HLA-A2, two subtypes of HLA-Bw60, two subtypes of HLA-Bw62, and four subtypes of HLA-B35 in a panel of 24 cells selected for the expression of HLA-B27. We envision that this technique will allow a rigorous classification of HLA-A,B antigens into novel subtypes. Populations of distinct ethnic origin are especially of interest in this regard. This technique might also be used as an additional criterion for the official classification of HLA-A,B antigens.

HLA Antigens↗
PubMed1990

Characterization of three human monoclonal antibodies specific for polymorphic class I and class II HLA antigens.

Three human monoclonal antibodies were derived from a single polytransfused patient awaiting renal transplantation. In microcitotoxicity assays, the patient's serum displayed strong positive reactions against greater than 90% of a panel of cells representing the known HLA specificities. The donor's peripheral blood lymphocytes were infected with Epstein-Barr virus, cloned, and supernatants of the virus transformed cultures were screened for the presence of IgG antilymphocyte reactivity utilizing an enzyme-linked immunosorbent assay method. Positive cultures were recloned and fused with the human-mouse heteromyeloma SHM. Supernatants from three clones were selected for alloreactivity and characterized by indirect immunofluorescent staining and fluoroactivated cell sorter analysis on homozygous typing cells, including those from the Tenth International Histocompatibility Workshop core panel and on cell lines derived from selected families. Data obtained demonstrate that two human monoclonal antibodies have DQw1 specificity, one of them being reactive against several DQw7-positive cell lines, while one monoclonal antibody is specific for the A2 + A28 class I MHC antigens. Anti-DQw1 antibodies were of different light-chain subtypes.

Antibodies, Monoclonal↗
PubMed1995

Studies on IgA class circulatory red cell antibodies--II. Properties of IgA class anti-A,anti-B.

The properties of IgA class red cell alloantibodies were determined using an anti-A,anti-B preparation confirmed to be free from IgG and IgM. The following characteristics were found: a thermal optimum of 37 degrees C; saline agglutination enhanced by anti-human IgA; activity substantially unchanged after 1 year's storage at 4 degrees C, but reduced by papain and destroyed by pepsin and by reduction and alkylation; neutralization by AB blood group substance; an inability to activate complement; and a distinctive pattern of apparent molecular sizes (reflecting dimeric and monomeric forms and complexes with serum proteins) which was significantly altered by the action of enzymes and by reduction and alkylation.

ABO Blood-Group System↗
PubMed1995

Studies on IgA class circulatory red cell antibodies--I. Preparation and identification of IgA class anti-A,anti-B.

To elucidate the role of IgA class alloantibodies in transfusion practice, anti-A,anti-B was prepared using pooled serum from healthy group O donors. IgG was removed by protein A and protein G, whilst IgM was extracted by concanavalin A and anti-human mu chain. The final preparation contained both IgA1 and IgA2; it had an IgA concentration similar to the original serum pool and an environment as near to normal as possible; the absence of IgG and IgM was confirmed by sensitive enzyme-linked antiglobulin tests, and extensive haemagglutination studies showed that anti-A,anti-B was the only red cell antibody present.

ABO Blood-Group System↗
PubMed2004

Chromophoric spin-labeled beta-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C beta-lactamases.

The chromophoric spin-label substrate 6-N-[3-(2,2,5,5-tetramethyl-1-oxypyrrolin-3-yl)-propen-2-oyl]penicillanic acid (SLPPEN) was synthesized by acylation of 6-aminopenicillanic acid with the acid chloride of 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propenoic acid and characterized by physical methods. By application of angle-selected electron nuclear double resonance (ENDOR), we have determined the molecular structure of SLPPEN in solution. SLPPEN exhibited UV absorption properties that allowed accurate monitoring of the kinetics of its enzyme-catalyzed hydrolysis. The maximum value of the (substrate-product) difference extinction coefficient was 2824 M(-1) cm(-1) at 275 nm compared to 670 M(-1) cm(-1) at 232 nm for SLPEN [J. Am. Chem. Soc. 117 (1995) 6739]. For SLPPEN, the steady-state kinetic parameters kcat and kcat/KM, determined under initial velocity conditions, were 637 +/- 36 s(-1) and 13.8 +/- 1.4 x 10(6) M(-1) s(-1), respectively, for hydrolysis catalyzed by TEM-1 beta-lactamase of E. coli, and 0.5 +/- 0.04 s(-1) and 3.9 +/- 0.4 x 10(4) M(-1) s(-1) for hydrolysis catalyzed by the beta-lactamase of Enterobacter cloacae P99. We have also observed "burst kinetics" for the hydrolysis of SLPPEN with P99 beta-lactamase, indicative of formation of an acylenzyme reaction intermediate. In DMSO:H2O (30:70, v:v) cryosolvent mixtures buffered to pH* 7.0, the half-life of the acylenzyme intermediate formed with the P99 enzyme at -5 degrees C was > or = 3 min, suitable for optical characterization. The observation of burst kinetics in the hydrolysis of SLPPEN catalyzed by P99 beta-lactamase suggests that this chromophoric spin-labeled substrate is differentially sensitive to active site interactions underlying the cephalosporinase and penicillinase reactivity of this class C enzyme.

Acylation↗
PubMed2006

Double loading of dendritic cell MHC class I and MHC class II with an AML antigen repertoire enhances correlates of T-cell immunity in vitro via amplification of T-cell help.

Therapeutic vaccination with dendritic cells presenting tumor-specific antigens is now recognized as an important investigational therapy for the treatment of neoplastic disease. Dendritic cell cross-presentation is credited with the ability of tumor lysate-loaded dendritic cells to prime both CD4 and CD8-specific T-lymphocyte responses, enabling the generation of cancer specific CTL activity without the loading of the classical MHC class I compartment. Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have doubly-loaded human dendritic cells with both AML-specific tumor lysate and AML-specific tumor mRNA. Our results show that these doubly-loaded dendritic cells can mediate superior primary, recall, and effector lytic responses in vitro in comparison to those of dendritic cells loaded with either tumor lysate or tumor mRNA alone. Enhanced recall responses appeared to be influenced by CD40/CD40L signaling, underscoring the importance of T-cell help in the generation and perpetuation of the adaptive immune response.

Antigens, Neoplasm↗
PubMed1993

Clinical evaluation of the use-dependent QRS prolongation and the reverse use-dependent QT prolongation of class I and class III antiarrhythmic agents and their value in predicting efficacy.

We measured the QRS duration during a treadmill exercise test and the QT interval using a 24-hour Holter electrocardiogram at various heart rates to identify use-dependent QRS prolongation and reverse use-dependent QT prolongation of class I and III antiarrhythmic drugs. Use-dependent QRS prolongation was detected in 61%, 53%, and 64% of patients receiving disopyramide, mexiletine, and pilsicainide, respectively. Reverse use-dependent QT prolongation was found in 40% and 70% of patients receiving disopyramide and E4031. Drugs suppressed > or = 75% of the total premature ventricular contractions in all patients who had both use-dependent QRS prolongation and reverse use-dependent QT prolongation, in 79% of patients with use-dependent QRS prolongation alone, in 70% with reverse use-dependent QT prolongation alone, and in 11% with neither use-dependent QRS prolongation nor reverse use-dependent QT prolongation. Use-dependent QRS prolongation and reverse use-dependent QT prolongation were identified noninvasively and were useful in evaluating antiarrhythmic efficacy.

Adult↗
PubMed1986

Separation of phospholipids by high-performance liquid chromatography: all major classes, including ethanolamine and choline plasmalogens, and most minor classes, including lysophosphatidylethanolamine.

High-performance liquid chromatographic methods for the separation and quantitation of phospholipids were developed and shown to give sensitive, reliable measurements of tissue phospholipids, including difficult-to-resolve pairs such as choline plasmalogen (plasmenylcholine) and phosphatidylcholine, choline glycerophospholipids and sphingomyelin, phosphatidylinositol and phosphatidylserine, and phosphatidylserine and lysophosphatidylethanolamine. Separations of most phospholipids including those mentioned above are more complete than in existing procedures, and require only 40 min per injection. Utilization of the hexane-2-propanol-water system has an advantage over separation techniques that employ acidic solvents in that the plasmalogens are not hydrolyzed and a less degradative environment for labile lipids is provided. Further, a rapid high-performance liquid chromatographic procedure for the separation of intact ethanolamine plasmalogen (plasmenylethanolamine) from phosphatidylethanolamine was developed. Previous procedures have required derivatized samples or acid hydrolysis of the plasmalogen vinyl ether linkage. A slight modification of the primary method (method I) increases the resolution of lysophosphatidylethanolamine from other classes (method II). A third modification (method III) can replace the standard silicic acid column separation of lipids into neutral, glycolipid, and phospholipid fractions.

Animals↗
PubMed2004

Calculation of the ground and excited states of a mixed valence compound [Fe2(OH)3(NH3)6]2+: a class II or class III compound?

The effective group potentials (EGP) approach has been successfully used for the computation of the ground and excited states energies of the mixed valence compound [Fe2(OH)3(NH3)6]2+. It is the first time that for a system as big as the complex presented above the ground and excited states are computed with their own orbitals and studied in such a detailed way. First of all, the NH3 EGP was validated by comparing calculations where NH3 was treated explicitly at different levels of calculations. Once the validation was obtained, the complete spectrum of the compound under interest was calculated and compared with results obtained in a previous work by Barone et al. and the spin Hamiltonian of widespread use. Some deviations from these predictive approaches were observed. This allowed us to emphasize the importance of the dynamic correlation which is not included explicitly in the spin Hamiltonian. Then, the influence of vibration has been studied by computing the potential energy curves obtained when moving the (OH)3 plane. This study shows that our calculations lead to a delocalized compound (class III) as expected according to former experimental data.

Journal Article↗
PubMed1990

Preferential over-expression of the class alpha rat Ya2 glutathione S-transferase subunit in livers bearing aflatoxin-induced pre-neoplastic nodules. Comparison of the primary structures of Ya1 and Ya2 with cloned class alpha glutathione S-transferase cDNA sequences.

Normal rat liver expresses Ya (Mr 25,500), Yc (Mr 27,500) and Yk (Mr 25,000) Class Alpha glutathione S-transferase (GST) subunits. The Ya-type subunit can be resolved into two separate polypeptides, designated Ya1 and Ya2, by reverse-phase h.p.l.c. In rat livers that possess aflatoxin B1-induced pre-neoplastic nodules, a marked increase is observed in the expression of Ya1, Ya2, Yc and Yk; of these subunits, Ya2 exhibited the greatest increase in concentration. The Ya1 and Ya2 subunits isolated from nodule-bearing livers were cleaved with CNBr, and the purified peptides were subjected to automated amino-acid-sequence analysis. Differences in the primary structures of the two Ya GST subunits were found at positions 31, 34, 107 and 117. These data demonstrate that Ya1 and Ya2 are distinct polypeptides and are the products of separate genes. The amino acid sequences obtained from Ya1 and Ya2 were compared with the cloned cDNAs pGTB 38 [Pickett, Telakowski-Hopkins, Ding, Argenbright & Lu (1984) J. Biol. Chem. 259, 4112-4115] and pGTR 261 [Lai, Li, Weiss, Reddy & Tu (1984) J. Biol. Chem. 259, 5182-5188], which encode rat Ya-type subunits. From these comparisons it appears probable that Ya1 represents the GST subunit encoded by pGTR 261, whereas Ya2 represents the subunit encoded by pGTB 38. It is likely that the over-expression of Ya1 and Ya2 in nodule-bearing livers is of major significance in the acquired resistance of nodules to aflatoxin B1, since previous work [Coles, Meyer, Ketterer, Stanton & Garner (1985) Carcinogenesis 6, 693-697] has shown that the Ya-type GST subunit has high activity towards aflatoxin B1 8,9-epoxide.

Aflatoxins↗
PubMed1997

Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse. Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human.

The cDNA encoding human glutathione S-transferase (GST) T1 has been expressed as two recombinant forms in Escherichia coli that could be purified by affinity chromatography on either IgG-Sepharose or nickel-agarose; one form of the transferase was synthesized from the pALP 1 expression vector as a Staphylococcus aureus protein A fusion, whereas the other form was synthesized from the pET-20b expression vector as a C-terminal polyhistidine-tagged recombinant. The yields of the two purified recombinant proteins from E. coli cultures were approx. 15 mg/l for the protein A fusion and 25 mg/l for the C-terminal polyhistidine-tagged GST T1-1. The purified recombinant proteins were catalytically active, although the protein A fusion was typically only 5-30% as active as the histidine-tagged GST. Both recombinant forms could catalyse the conjugation of glutathione with the model substrates 1,2-epoxy-3-(4'-nitrophenoxy)propane,4-nitrobenzyl chloride and 4-nitrophenethyl bromide but were inactive towards 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 1-menaphthyl sulphate. Recombinant human GST T1-1 was found to exhibit glutathione peroxidase activity and could catalyse the reduction of cumene hydroperoxide. In addition, recombinant human GST T1-1 was found to conjugate glutathione with dichloromethane, a pulmonary and hepatic carcinogen in the mouse. Immunoblotting with antibodies raised against different transferase isoenzymes showed that GST T1-1 is expressed in a large number of human organs in a tissue-specific fashion that differs from the pattern of expression of classes Alpha, Mu and Pi GST. Most significantly, GST T1-1 was found in only low levels in human pulmonary soluble extract of cells, suggesting that in man the lung has little capacity to activate the volatile dichloromethane.

Amino Acid Sequence↗