Treating asthma with omega-3 fatty acids: where is the evidence? A systematic review.

BACKGROUND: Considerable interest exists in the potential therapeutic value of dietary supplementation with the omega-3 fatty acids. Given the interplay between pro-inflammatory omega-6 fatty acids, and the less pro-inflammatory omega-3 fatty acids, it has been thought that the latter could play a key role in treating or preventing asthma. The purpose was to systematically review the scientific-medical literature in order to identify, appraise, and synthesize the evidence for possible treatment effects of omega-3 fatty acids in asthma. METHODS: Medline, Premedline, Embase, Cochrane Central Register of Controlled Trials, CAB Health, and, Dissertation Abstracts were searched to April 2003. We included randomized controlled trials (RCT's) of subjects of any age that used any foods or extracts containing omega-3 fatty acids as treatment or prevention for asthma. Data included all asthma related outcomes, potential covariates, characteristics of the study, design, population, intervention/exposure, comparators, and co interventions. RESULTS: Ten RCT's were found pertinent to the present report. CONCLUSION: Given the largely inconsistent picture within and across respiratory outcomes, it is impossible to determine whether or not omega-3 fatty acids are an efficacious adjuvant or monotherapy for children or adults. Based on this systematic review we recommend a large randomized controlled study of the effects of high-dose encapsulated omega-3 fatty acids on ventilatory and inflammatory measures of asthma controlling diet and other asthma risk factors. This review was limited because Meta-analysis was considered inappropriate due to missing data; poorly or heterogeneously defined populations, interventions, intervention-comparator combinations, and outcomes. In addition, small sample sizes made it impossible to meaningfully assess the impact on clinical outcomes of co-variables. Last, few significant effects were found.

Structural requirements for Arabidopsis beta1,2-xylosyltransferase activity and targeting to the Golgi.

Characterization of a beta1,2-xylosyltransferase from Arabidopsis thaliana (AtXylT) was carried out by expression in Sf9 insect cells using a baculovirus vector system. Serial deletions at both the N- and C-terminal ends proved that integrity of a large domain located between amino acid 31 and the C-terminal lumenal region is required for AtXylT activity expression. The influence of N-glycosylation on AtXylT activity has been evaluated using either tunicamycin or mutagenesis of potential N-glycosylation sites. AtXylT is glycosylated on two of its three potential N-glycosylation sites (Asn51, Asn301, Asn478) and the occupancy of at least one of these two sites (Asn51 and Asn301) is necessary for AtXylT stability and activity. Contribution of the N-terminal part of AtXylT in targeting and intracellular distribution of this protein was studied by expression of variably truncated, GFP-tagged AtXylT forms in tobacco cells using confocal and electron microscopy. These studies have shown that the transmembrane domain of AtXylT and its short flanking amino acid sequences are sufficient to specifically localize a reporter protein to the medial Golgi cisternae in tobacco cells. This study is the first detailed characterization of a plant glycosyltransferase at the molecular level.

Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.

UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a medial-Golgi enzyme that is essential for the processing of oligomannose to hybrid and complex N-glycans. On the basis of highly conserved sequences obtained from previously cloned mammalian GnTI genes, cDNAs for two closely related GnTI isoenzymes were isolated from a Xenopus laevis ovary cDNA library. As typical for glycosyltransferases, both proteins exhibit a type II transmembrane protein topology with a short N-terminal cytoplasmic tail (4 amino acids); a transmembrane domain of 22 residues; a stem region with a length of 81 (isoenzyme A) and 77 (isoenzyme B) amino acids, respectively; and a catalytic domain consisting of 341 residues. The two proteins differ not only in length but also at 13 (stem) and 18 (catalytic domain) positions, respectively. The overall identity of the catalytic domains of the X. laevis GnTI isoenzymes with their mammalian and plant orthologues ranges from 30% (Nicotiana tabacum) to 67% (humans). Isoenzymes A and B are encoded by two separate genes that were both found to be expressed in all tissues examined, albeit in varying amounts and ratios. On expression of the cDNAs in the baculovirus/insect cell system, both isoenzymes were found to exhibit enzymatic activity. Isoenzyme B is less efficiently folded in vivo and thus appears of lower physiological relevance than isoenzyme A. However, substitution of threonine at position 223 with alanine was sufficient to confer isoenzyme B with properties similar to those observed for isoenzyme A.

Congenital disorders involving defective N-glycosylation of proteins.

This review deals with several of the main autosomal recessive congenital disorders involving defective N-glycosylation of proteins (the addition of glycans linked to the polypeptide chain by a beta-linkage between the anomeric carbon of N-acetylglucosamine and the amido group of L-asparagine). These congenital disorders of glycosylation (CDG, previously known as carbohydrate-deficient glycoprotein syndromes) are a group of multisystemic diseases often involving severe psychomotor retardation. Six distinct variants of CDG in group I (types Ia-If) have been described to date and the defects have been localized to deficiencies in the assembly of the dolichylpyrophosphate-linked oligosaccharide N-glycan precursor and its transfer to asparagine residues on the nascent polypeptides. Two variants of CDG group II (types IIa and IIb) have been identified as defects in the processing of protein-bound N-glycans. Hereditary erythroblastic multinuclearity with a positive acidified-serum lysis test (HEMPAS; congenital dyserythropoietic anemia type II) presents as a relatively mild dyserythropoietic anemia. The genetic defect in most cases of HEMPAS is not known, but alpha-3/6-mannosidase II is involved in at least some patients. Leukocyte adhesion deficiency type II (LAD II) is a rare disorder characterized by recurrent infections, persistent leukocytosis and severe mental and growth retardation. LAD II is due to lack of availability of GDP-fucose. The study of these diseases and of relevant animal models has provided strong evidence that N-glycans are essential for normal mammalian development.

Glycoconjugate abnormalities in patients with congenital dyserythropoietic anaemia type I, II and III.

Congenital dyserythropoietic anaemia type II (CDA II) is well known for glycosylation abnormalities affecting erythrocyte membrane glycoconjugates that encompass hypoglycosylation of band 3 glycoprotein and accumulation of glycosphingolipids: lactotriaosylceramides, neolactotriaosylceramide and polyglycosylceramides. These abnormalities were not observed in erythrocytes from patients with CDA of either type I or III. Recently, however, we have described a CDA type I patient in Poland with identical, though less pronounced, glycoconjugate abnormalities to those observed in patients with CDA type II. The abnormalities included partial unglycosylation of O-linked glycosylation sites in glycophorin A. These abnormalities are now reported in three Bedouin patients from Israel with CDA type I. In addition, the erythrocyte membranes of these patients exhibited highly increased globotetraosylceramide content. Glycoconjugate abnormalities were also present in erythrocyte membranes from three patients from Northern Sweden with CDA type III but they almost exclusively affected glycosphingolipids. In erythrocytes of all patients examined including one with CDA type II, polyglycosylceramides were significantly hypoglycosylated although, on a molar basis, their contents in erythrocyte membranes were increased. Thus, glycoconjugate abnormalities of varying intensity occur in erythrocyte membranes from all patients with CDA that were investigated.

Cloning and expression of Drosophila melanogaster UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in lambda-ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of cDNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-terminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the N-terminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an N-terminal (His)6 tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta-octyl (M3-octyl) to Man alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta-octyl. The Km values (0.7 and 0.03 mM for M3-octyl and UDP-GlcNAc respectively), temperature optimum (37 degrees C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.

Modeling human congenital disorder of glycosylation type IIa in the mouse: conservation of asparagine-linked glycan-dependent functions in mammalian physiology and insights into disease pathogenesis.

The congenital disorders of glycosylation (CDGs) are recent additions to the repertoire of inherited human genetic diseases. Frequency of CDGs is unknown since most cases are believed to be misdiagnosed or unrecognized. With few patients identified and heterogeneity in disease signs noted, studies of animal models may provide increased understanding of pathogenic mechanisms. However, features of mammalian glycan biosynthesis and species-specific variations in glycan repertoires have cast doubt on whether animal models of human genetic defects in protein glycosylation will reproduce pathogenic events and disease signs. We have introduced a mutation into the mouse germline that recapitulates the glycan biosynthetic defect responsible for human CDG type IIa (CDG-IIa). Mice lacking the Mgat2 gene were deficient in GlcNAcT-II glycosyltransferase activity and complex N-glycans, resulting in severe gastrointestinal, hematologic, and osteogenic abnormalities. With use of a lectin-based diagnostic screen for CDG-IIa, we found that all Mgat2-null mice died in early postnatal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors. Mice deficient in complex N-glycans exhibited most CDG-IIa disease signs; however, some signs were unique to the aged mouse or are prognostic in human CDG-IIa. Unexpectedly, analyses of N-glycan structures in Mgat2-null mice revealed a novel oligosaccharide branch on the "bisecting" N-acetylglucosamine. These genetic, biochemical, and physiologic studies indicate conserved functions for N-glycan branches produced in the Golgi apparatus among two mammalian species and suggest possible therapeutic approaches to GlcNAcT-II deficiency. Our findings indicate that human genetic disease due to aberrant protein glycosylation can be modeled in the mouse to gain insights into N-glycan-dependent physiology and the pathogenesis of CDG-IIa.

How efficacious and safe is short-acting methylphenidate for the treatment of attention-deficit disorder in children and adolescents? A meta-analysis.

BACKGROUND: Numerous small clinical trials have been carried out to study the behaviourally defined efficacy and safety of short-acting methylphenidate compared with placebo for attention-deficit disorder (ADD) in individuals aged 18 years and less. However, no meta-analyses that carefully examined these questions have been done. We reviewed the behavioural evidence from all the randomized controlled trials that compared methylphenidate and placebo, and completed a meta-analysis. METHODS: We searched several electronic sources for articles published between 1981 and 1999: MEDLINE, EMBASE, PsychINFO, ERIC, CINAHL, HEALTHSTAR, Biological Abstracts, Current Contents and Dissertation Abstracts. The Cochrane Library Trials Registry and Current Controlled Trials were also consulted. A study was considered eligible for inclusion if it entailed the following: a placebo-controlled randomized trial that involved short-acting methylphenidate and participants aged 18 years or less at the start of the trial who had received any primary diagnosis of ADD that was made in a systematic and reproducible way. RESULTS: We included 62 randomized trials that involved a total of 2897 participants with a primary diagnosis of ADD (e.g., with or without hyperactivity). The median age of trial participants was 8.7 years, and the median "percent male" composition of trials was 88.1%. Most studies used a crossover design. Using the scores from 2 separate indices, this collection of trials exhibited low quality. Interventions lasted, on average, 3 weeks, with no trial lasting longer than 28 weeks. Each primary outcome (hyperactivity index) demonstrated a significant effect of methylphenidate (effect size reported by teacher 0.78, 95% confidence interval [CI] 0.64-0.91; effect size reported by parent 0.54, 95% CI 0.40-0.67). However, these apparent beneficial effects are tempered by a strong indication of publication bias and the lack of robustness of the findings, especially those involving core ADD features. Methylphenidate also has an adverse event profile that requires consideration. For example, clinicians only need to treat 4 children to identify an episode of decreased appetite. INTERPRETATION: Short-acting methylphenidate has a statistically significant clinical effect in the short-term treatment of individuals with a diagnosis of ADD aged 18 years and less. However, the extension of this placebo-controlled effect beyond 4 weeks of treatment has not been demonstrated. Exact knowledge of the extent and definition of the short-term behavioural usefulness of methylphenidate is questioned.

X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily.

N:-acetylglucosaminyltransferase I (GnT I) serves as the gateway from oligomannose to hybrid and complex N:-glycans and plays a critical role in mammalian development and possibly all metazoans. We have determined the X-ray crystal structure of the catalytic fragment of GnT I in the absence and presence of bound UDP-GlcNAc/Mn(2+) at 1.5 and 1.8 A resolution, respectively. The structures identify residues critical for substrate binding and catalysis and provide evidence for similarity, at the mechanistic level, to the deglycosylation step of retaining beta-glycosidases. The structuring of a 13 residue loop, resulting from UDP-GlcNAc/Mn(2+) binding, provides an explanation for the ordered sequential 'Bi Bi' kinetics shown by GnT I. Analysis reveals a domain shared with Bacillus subtilis glycosyltransferase SpsA, bovine beta-1,4-galactosyl transferase 1 and Escherichia coli N:-acetylglucosamine-1-phosphate uridyltransferase. The low sequence identity, conserved fold and related functional features shown by this domain define a superfamily whose members probably share a common ancestor. Sequence analysis and protein threading show that the domain is represented in proteins from several glycosyltransferase families.

Regulation of expression of the human beta-1,2-N-acetylglucosaminyltransferase II gene (MGAT2) by Ets transcription factors.

Oncogenic transformation of fibroblasts by the src oncogene has long been known to cause an increase in the size of cell-surface protein-bound oligosaccharides, owing primarily to increased N-glycan branching mediated by increased beta-1,6-N-acetylglucosaminyltransferase V (GnT V) activity. The src-responsive element of the GnT V promoter was localized to Ets-binding sites and the promoter was transcriptionally stimulated by both ets-1 and ets-2 expression [Buckhaults, Chen, Fregien and Pierce (1997) J. Biol. Chem. 272, 19575-19581; Kang, Saito, Ihara, Miyoshi, Koyama, Sheng and Taniguchi (1996) J. Biol. Chem. 271, 26706-26712]. Because GnT V action requires the prior action of beta-1,2-N-acetylglucosaminyltransferase II (GnT II) and the human GnT II promoter contains four putative Ets-binding sites [Chen, Zhou, Tan and Schachter (1998) Glycoconj. J. 15, 301-308], GnT II might also be under oncogenic control via Ets transcription factors. We now report that co-transfection into HepG2 or COS-1 cells of either ets-1 or ets-2 expression plasmids together with chimaeric GnT II promoter-chloramphenicol acetyltransferase plasmids results in a 2-4-fold stimulation of promoter activity. Mobility-shift assays and South-Western blots localized the functional Ets-binding site to one of the four putative sites on the GnT II promoter. The GnT II promoter, unlike the GnT V promoter, is not activated by either src or neu. Therefore although both promoters are stimulated by a member of the Ets family of transcription factors, the functional role of this Ets transcriptional control seems to be different for the two genes.

Molecular cloning and expression analysis of a mouse UDP-GlcNAc:Gal(beta1-4)Glc(NAc)-R beta1,3-N-acetylglucosaminyltransferase homologous to Drosophila melanogaster Brainiac and the beta1,3-galactosyltransferase family.

We have isolated a murine cDNA coding for a beta1,3-N-acetylglucosaminyltransferase enzyme ( beta3GnT). This enzyme is similar in sequence to Drosophila melanogaster Brainiac and to the murine and human beta1,3-galactosyltransferase family of proteins. The mouse beta 3GnT protein is 397 amino acids in length and contains 7 cysteine residues that are conserved in the human orthologue. beta 3GnT is a type II membrane protein localized to the Golgi apparatus. Enzyme assays with recombinant mouse beta 3GnT reveal that it has a preference for acceptors with Gal(beta1-4)Glc(NAc) at the non-reducing termini. Proton NMR analysis of product showed incorporation of GlcNAc in beta1,3 linkage to the terminal Gal of Gal(beta1-4)Glc(beta1-O-benzyl). Northern blot analysis revealed the presence of a single 3.0[emsp4 ]kb transcript in all adult mouse and human organs tested, with highest levels in the kidney, liver, heart and placenta. The beta 3GnT gene is also expressed in a number of tumor cell lines. The human orthologue of beta 3GnT is located on chromosome 2pl5.

The joys of HexNAc. The synthesis and function of N- and O-glycan branches.

This review covers discoveries made over the past 30-35 years that were important to our understanding of the synthetic pathway required for initiation of the antennae or branches on complex N-glycans and O-glycans. The review deals primarily with the author's contributions but the relevant work of other laboratories is also discussed. The focus of the review is almost entirely on the glycosyltransferases involved in the process. The following topics are discussed. (1) The localization of the synthesis of complex N-glycan antennae to the Golgi apparatus. (2) The "evolutionary boundary" at the stage in N-glycan processing where there is a change from oligomannose to complex N-glycans; this switch correlates with the appearance of multicellular organisms. (3) The discovery of the three enzymes which play a key role in this switch, N-acetylglucosaminyltransferases I and II and mannosidase II. (4) The "yellow brick road" which leads from oligomannose to highly branched complex N-glycans with emphasis on the enzymes involved in the process and the factors which control the routes of synthesis. (5) A short discussion of the characteristics of the enzymes involved and of the genes that encode them. (6) The role of complex N-glycans in mammalian and Caenorhabditis elegans development. (7) The crystal structure of N-acetylglucosaminyltransferase I. (8) The discovery of the enzymes which synthesize O-glycan cores 1, 2, 3 and 4 and their elongation.

Carbohydrate-deficient glycoprotein syndrome type II.

The carbohydrate-deficient glycoprotein syndromes (CDGS) are a group of autosomal recessive multisystemic diseases characterized by defective glycosylation of N-glycans. This review describes recent findings on two patients with CDGS type II. In contrast to CDGS type I, the type II patients show a more severe psychomotor retardation, no peripheral neuropathy and a normal cerebellum. The CDGS type II serum transferrin isoelectric focusing pattern shows a large amount (95%) of disialotransferrin in which each of the two glycosylation sites is occupied by a truncated monosialo-monoantennary N-glycan. Fine structure analysis of this glycan suggested a defect in the Golgi enzyme UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) which catalyzes an essential step in the biosynthetic pathway leading from hybrid to complex N-glycans. GnT II activity is reduced by over 98% in fibroblast and mononuclear cell extracts from the CDGS type II patients. Direct sequencing of the GnT II coding region from the two patients identified two point mutations in the catalytic domain of GnT II, S290F (TCC to TTC) and H262R (CAC to CGC). Either of these mutations inactivates the enzyme and probably also causes reduced expression. The CDG syndromes and other congenital defects in glycan synthesis as well as studies of null mutations in the mouse provide strong evidence that the glycan moieties of glycoproteins play essential roles in the normal development and physiology of mammals and probably of all multicellular organisms.