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1.
Hum Mol Genet ; 19(5): 920-30, 2010 Mar 01.
Article in English | MEDLINE | ID: mdl-20015954

ABSTRACT

Mammals and birds have common embryological facial structures, and appear to employ the same molecular genetic developmental toolkit. We utilized natural variation found in bird beaks to investigate what genes drive vertebrate facial morphogenesis. We employed cross-species microarrays to describe the molecular genetic signatures, developmental signaling pathways and the spectrum of transcription factor (TF) gene expression changes that differ between cranial neural crest cells in the developing beaks of ducks, quails and chickens. Surprisingly, we observed that the neural crest cells established a species-specific TF gene expression profile that predates morphological differences between the species. A total of 232 genes were differentially expressed between the three species. Twenty-two of these genes, including Fgfr2, Jagged2, Msx2, Satb2 and Tgfb3, have been previously implicated in a variety of mammalian craniofacial defects. Seventy-two of the differentially expressed genes overlap with un-cloned loci for human craniofacial disorders, suggesting that our data will provide a valuable candidate gene resource for human craniofacial genetics. The most dramatic changes between species were in the Wnt signaling pathway, including a 20-fold up-regulation of Dkk2, Fzd1 and Wnt1 in the duck compared with the other two species. We functionally validated these changes by demonstrating that spatial domains of Wnt activity differ in avian beaks, and that Wnt signals regulate Bmp pathway activity and promote regional growth in facial prominences. This study is the first of its kind, extending on previous work in Darwin's finches and provides the first large-scale insights into cross-species facial morphogenesis.


Subject(s)
Avian Proteins/metabolism , Birds/embryology , Birds/genetics , Gene Expression Regulation, Developmental , Transcription Factors/metabolism , Animals , Avian Proteins/genetics , Beak/embryology , Body Patterning , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Cell Differentiation , Chick Embryo , Chickens/metabolism , Craniofacial Abnormalities/genetics , Embryo, Nonmammalian/metabolism , Gene Expression Profiling , Humans , Morphogenesis , Transcription Factors/genetics , Wnt Proteins/genetics , Wnt Proteins/metabolism
2.
Otol Neurotol ; 22(2): 195-9, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11300268

ABSTRACT

OBJECTIVE: To compare surgical experience and measures of electrode and patient performance of children who were implanted with the Clarion (Advanced Bionics, Sylmar, CA, U.S.A.) device with and without the new electrode positioner (EP). STUDY DESIGN: Prospectively and retrospectively collected data were compared between two independent groups. SETTING: Tertiary care children's hospital. PATIENTS: Twenty-four children (mean age, 3.0 years) implanted during the original Food and Drug Administration (FDA) clinical trial required for commercial approval of the Clarion and 15 children (mean age, 3.4 years) implanted with the EP as part of an ongoing FDA trial. INTERVENTION: Cochlear implant with and without EP. MAIN OUTCOME MEASURES: Electrical psychophysical threshold, most comfortable loudness level (MCL), electrode impedance, and speech perception measures were compared at 3 and 6 months after initial stimulation. RESULTS AND CONCLUSION: All children had complete insertion of electrodes. No difficulty inserting the EP occurred nor did subsequent related complications. Subjects with the EP had significantly lower threshold and MCL levels. Electrode impedance declined on stimulated electrodes in both groups. Meaningful Auditory Integration Scale scores significantly improved in both groups; the EP group appeared to receive as much benefit as the non-EP group.


Subject(s)
Cochlear Implantation , Hearing Loss, Sensorineural/surgery , Auditory Threshold/physiology , Child, Preschool , Humans , Infant , Preoperative Care , Prospective Studies , Psychophysics , Retrospective Studies , Speech Perception/physiology
3.
J Biol Chem ; 276(16): 12785-90, 2001 Apr 20.
Article in English | MEDLINE | ID: mdl-11278878

ABSTRACT

The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha-linked galactose from a P(k) epitope is substituted at the O6 position (Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626-633). Using a synthetic P(k)-epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E(L1)-derived sialyltransferase can make both alpha-2,3 and alpha-2,6 linkages to the terminal galactose. Gene disruption experiments showed that the lst gene in 126E(L1) was responsible for the in vivo addition of the alpha-2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) alpha-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) alpha-2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild type 126E(L1), indicating that residue 168 is the critical residue for the alpha-2,6-sialyltransferase activity in vitro as well as in vivo.


Subject(s)
Lipopolysaccharides/biosynthesis , Lipopolysaccharides/chemistry , Neisseria meningitidis/enzymology , Sialyltransferases/chemistry , Sialyltransferases/metabolism , Amino Acid Substitution , Carbohydrate Sequence , Glycosides/biosynthesis , Glycosides/chemistry , Glycosyltransferases/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Neisseria meningitidis/classification , Neisseria meningitidis/immunology , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , beta-D-Galactoside alpha 2-6-Sialyltransferase , beta-Galactoside alpha-2,3-Sialyltransferase
4.
J Biol Chem ; 275(47): 37006-10, 2000 Nov 24.
Article in English | MEDLINE | ID: mdl-10913148

ABSTRACT

The complex of Maclura pomifera agglutinin with the T-antigen disaccharide (beta-d-Gal-(1-->3)-alpha-d-GalNAc-(1-->O)-Me) was investigated by NMR spectroscopy in aqueous solution. Intramolecular transferred nuclear Overhauser enhancement (NOE) effects between the monosaccharide moieties were used to derive the ligand conformation in the lectin-bound state. Ligand protons in contact with the protein were identified by saturation transfer difference experiments and intermolecular transferred NOE effects. It is demonstrated that structural differences exist for the ligand-lectin complex in aqueous solution as compared with the previously published crystal structure (Lee, X., Thompson, A., Zhiming, Z., Ton-that, H., Biesterfeldt, J., Ogata, C., Xu, L., Johnston, R. A. Z. , and Young, N. M. (1998) J. Biol. Chem. 273, 6312-6318). In order to accommodate the O-methyl group of the disaccharide, the amino acid side chain of Tyr-122 has to rotate from its position in the crystal. The NMR data are in accord with two conformational families at the beta-(1-->3)glycosidic linkage in the solution complex with interglycosidic angles phi/psi = 45/-65 degrees and -65/-18 degrees. These differ from the bound conformation of the ligand in the crystal (phi/psi = 39/-8 degrees ) and are not highly populated by the ligand in the free state. The reason for the structural differences at the beta-(1-->3)glycosidic linkage are hydrogen bonds that stabilize the relative orientation of the monosaccharide units in the crystal. Our results demonstrate that the crystallization of a protein-carbohydrate complex can interfere with the delicate process of carbohydrate recognition in solution.


Subject(s)
Antigens, Neoplasm/metabolism , Antigens, Tumor-Associated, Carbohydrate/metabolism , Lectins/metabolism , Plant Lectins , Antigens, Neoplasm/chemistry , Antigens, Tumor-Associated, Carbohydrate/chemistry , Biomarkers, Tumor , Carbohydrate Conformation , Lectins/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Binding , Protein Conformation
5.
J Biol Chem ; 275(6): 3896-906, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660542

ABSTRACT

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS.


Subject(s)
Campylobacter jejuni/enzymology , Gangliosides/biosynthesis , Glycosyltransferases/genetics , Amino Acid Sequence , Campylobacter Infections/microbiology , Campylobacter jejuni/genetics , Carbohydrate Sequence , Cloning, Molecular , Gangliosides/chemistry , Glycosyltransferases/chemistry , Guillain-Barre Syndrome/microbiology , Lipopolysaccharides/biosynthesis , Lipopolysaccharides/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Oligosaccharides/biosynthesis , Oligosaccharides/chemistry , Sequence Alignment , Sialyltransferases/chemistry , Sialyltransferases/genetics
6.
Otolaryngol Head Neck Surg ; 122(2): 183-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10652387

ABSTRACT

OBJECTIVE: The goals of this study were to retrospectively review high-resolution CTs (HRCTs) of pediatric postmeningitic cochlear implant recipients and to correlate results with surgical findings. METHODS: HRCTs of 20 children (11 months to 12 years old) who underwent implantation with multichannel devices were reviewed. Results were correlated with the degree of ossification observed at surgery. RESULTS: Ninety percent of subjects required drilling of ossified bone within the basal turn at surgery. HRCT of the cochleas suggested ossification within the basal turn in 45% (50% sensitivity). Ossification of the lateral semicircular canal on HRCT was present in 72% (77% sensitivity). Five of 6 cases without radiographic evidence of ossification had positive findings at surgery. CONCLUSION: Ossification is a common occurrence in postmeningitic deaf children. Ossification of the lateral semicircular canal on HRCT is a more sensitive measure for predicting ossification than evidence of cochlear involvement. Absence of ossification on HRCT is no guarantee of cochlear patency at the time of implantation.


Subject(s)
Cochlea/diagnostic imaging , Cochlear Implantation , Deafness/rehabilitation , Meningitis, Bacterial/complications , Ossification, Heterotopic/diagnostic imaging , Child , Child, Preschool , Cochlea/pathology , Deafness/diagnostic imaging , Deafness/etiology , Deafness/pathology , Female , Humans , Infant , Male , Ossification, Heterotopic/etiology , Ossification, Heterotopic/pathology , Retrospective Studies , Semicircular Canals/diagnostic imaging , Semicircular Canals/pathology , Sensitivity and Specificity , Tomography, X-Ray Computed
7.
Trans Am Ophthalmol Soc ; 98: 237-42; discussion 243-5, 2000.
Article in English | MEDLINE | ID: mdl-11190026

ABSTRACT

PURPOSE: To screen severe to profound, preverbal hearing-impaired children for Usher syndrome by ophthalmologic examinations, including electroretinographic testing. These patients are especially good candidates for early cochlear implants, which will improve listening and spoken language skills. METHODS: Consecutive patients over 2 years of age, given a diagnosis of severe to profound, preverbal hearing loss, were screened for Usher syndrome by a complete ophthalmologic examination including an electroretinogram. RESULTS: Five of 48 patients screened (10.4%) were diagnosed with Usher syndrome and received cochlear implants. CONCLUSION: All children with severe to profound, preverbal sensorineural hearing loss should be screened for Usher syndrome by ophthalmologic examination including electroretinogram.


Subject(s)
Genes, Recessive , Hearing Loss, Sensorineural/diagnosis , Hearing Loss, Sensorineural/genetics , Retinitis Pigmentosa/diagnosis , Retinitis Pigmentosa/genetics , Vision Disorders/diagnosis , Audiology , Child , Child, Preschool , Cochlear Implants , Electroretinography , Hearing Loss, Sensorineural/congenital , Hearing Loss, Sensorineural/surgery , Humans , Ophthalmology/methods , Physical Examination , Reflex, Vestibulo-Ocular , Retinitis Pigmentosa/complications , Syndrome , Vision Disorders/etiology
9.
Mol Immunol ; 36(6): 339-47, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10443998

ABSTRACT

The crystal structure of the complex of an anti-Id Fab with an Fab specific for a Brucella polysaccharide antigen has previously been reported (Evans et al., 1994, J. Mol. Biol. 241, 691-705). To complement this study, the binding characteristics and immunological properties of this Ab2 and two others raised with a second anti-Brucella antibody were investigated, including quantitative kinetic measurements by surface plasmon resonance. The affinities of the Fabs from the Ab2s for the Ab1s were three orders of magnitude greater than those estimated for the antigen, but the Ab2s failed to induce antigen-binding Ab3s, that is, they were of the Ab2gamma type. The avidities of the Ab1s for antigen were however within one order of magnitude of their avidities for Ab2. Tests of 16 other anti-Brucella polysaccharide antibodies showed that the two idiotopes were not present in them, and in confirmation of the lack of a dominant idiotope, N-terminal sequencing of their H and L chains showed a wide variety of V genes were employed in the immune response to the Brucella polysaccharides. The failure of the Ab2 to induce antigen-reactive Ab3 thus appears to be due to neither intrinsic affinity nor idiotope frequency, but arises instead from structural reasons, for example, the incomplete penetration of the Ab2 into the binding-site cleft of the Ab1. The surface topography of polysaccharide antigens and their binding-sites thus appears to be especially difficult for Ab2s to mimic and will restrict their routine use as surrogates for T-cell independent polysaccharide antigens.


Subject(s)
Antibodies, Bacterial/immunology , Brucella/immunology , Immunoglobulin Idiotypes/immunology , Polysaccharides, Bacterial/immunology , Amino Acid Sequence , Animals , Antibodies, Bacterial/chemistry , Enzyme-Linked Immunosorbent Assay , Female , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Idiotypes/chemistry , Mice , Mice, Inbred BALB C , Molecular Mimicry , Molecular Sequence Data , Polysaccharides, Bacterial/chemistry , Sequence Analysis , Sequence Homology, Amino Acid , Surface Plasmon Resonance
10.
Ann Otol Rhinol Laryngol Suppl ; 177: 99-103, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10214811

ABSTRACT

This study compares the auditory perceptual skill development of 23 congenitally deaf children who received the Nucleus 22-channel cochlear implant with the SPEAK speech coding strategy, and 20 children who received the CLARION Multi-Strategy Cochlear Implant with the Continuous Interleaved Sampler (CIS) speech coding strategy. All were under 5 years old at implantation. Preimplantation, there were no significant differences between the groups in age, length of hearing aid use, or communication mode. Auditory skills were assessed at 6 months and 12 months after implantation. Postimplantation, the mean scores on all speech perception tests were higher for the Clarion group. These differences were statistically significant for the pattern perception and monosyllable subtests of the Early Speech Perception battery at 6 months, and for the Glendonald Auditory Screening Procedure at 12 months. Multiple regression analysis revealed that device type accounted for the greatest variance in performance after 12 months of implant use. We conclude that children using the CIS strategy implemented in the Clarion implant may develop better auditory perceptual skills during the first year postimplantation than children using the SPEAK strategy with the Nucleus device.


Subject(s)
Cochlear Implants , Speech Perception/physiology , Child, Preschool , Cochlear Implantation , Deafness/congenital , Deafness/physiopathology , Deafness/rehabilitation , Deafness/surgery , Hearing Tests , Humans , Infant , Postoperative Period
11.
FEBS Lett ; 446(1): 203-6, 1999 Mar 05.
Article in English | MEDLINE | ID: mdl-10100643

ABSTRACT

Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed.


Subject(s)
Fabaceae/metabolism , Glycoproteins/biosynthesis , Lectins/biosynthesis , Plant Proteins/biosynthesis , Plants, Medicinal , Protein Processing, Post-Translational , Amino Acid Sequence , Enzyme Inhibitors , Fabaceae/genetics , Glycoproteins/genetics , Intercellular Signaling Peptides and Proteins , Lectins/genetics , Molecular Sequence Data , Plant Lectins , Plant Proteins/genetics , alpha-Amylases/antagonists & inhibitors
12.
Nat Biotechnol ; 16(8): 769-72, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9702777

ABSTRACT

Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitidis, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and alpha-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an alpha-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce alpha-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.


Subject(s)
Multienzyme Complexes/metabolism , N-Acylneuraminate Cytidylyltransferase/metabolism , Oligosaccharides/biosynthesis , Recombinant Fusion Proteins/metabolism , Sialyltransferases/metabolism , Catalysis , Chemical Precipitation , Chromatography, Affinity , Enzyme Stability , Escherichia coli/genetics , Hydrogen-Ion Concentration , Ion Exchange , Lactose/metabolism , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , N-Acetylneuraminic Acid/metabolism , N-Acylneuraminate Cytidylyltransferase/biosynthesis , N-Acylneuraminate Cytidylyltransferase/chemistry , N-Acylneuraminate Cytidylyltransferase/genetics , Neisseria meningitidis/enzymology , Neisseria meningitidis/genetics , Neuraminic Acids/metabolism , Phosphoenolpyruvate/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Sialyltransferases/biosynthesis , Sialyltransferases/chemistry , Solubility , Ultrafiltration , beta-Galactoside alpha-2,3-Sialyltransferase
13.
Protein Eng ; 11(4): 295-302, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9680191

ABSTRACT

The lgtB gene encoding a beta-1,4-galactosyltransferase gene and the lgtC gene encoding an alpha-1,4-galactosyltransferase from the bacterial pathogen Neisseria meningitidis were cloned into an expression vector and overexpressed in Escherichia coli. Both genes expressed very well, but problems with C-terminal proteolysis were encountered with both proteins. The lgtC protein was initially isolated from extracts of recombinant E.coli as a truncated species that retained enzymatic activity, and was subsequently shown by mass spectrometry to be 19 residues shorter than the expected protein. A specific set of engineered C-terminal deletions was constructed to investigate their effect on the expression of lgtC. As many as 28 residues could be deleted with little effect on activity, and with the concomitant improvement of the overall expression up to fivefold over the full length protein. The lgtB protein was also proteolysed in extracts of normal E.coli strains into enzymatically inactive fragments lacking 28 or 41 C-terminal residues. This degradation could be prevented by expression in an ompT protease deficient strain of E.coli. The full length lgtB protein was not stable in soluble protein extracts from all recombinant strains, however a stable enzyme preparation could be achieved with the membrane fraction from cells of the ompT deficient strain expressing lgtB. Specific deletions of lgtB were also constructed, and 15 residues could be removed without loss of enzyme activity and also with the concomitant improvement of the overall expression up to twofold over the full length protein. Longer deletions produced protein but activity could not be detected in these recombinant strains. Examination of the glycosyltransferase sequences from a wide range of bacteria showed their C-terminal segments of approximately 50 amino acids frequently contained paired basic residues. Engineering of these segments may therefore be required as a general practice to produce these enzymes for use in the large scale chemi-enzymatic synthesis of carbohydrate-based therapeutics.


Subject(s)
Galactosyltransferases/metabolism , Neisseria meningitidis/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Recombinant , Escherichia coli/genetics , Galactosyltransferases/chemistry , Kinetics , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sulfhydryl Compounds/metabolism
14.
J Biol Chem ; 273(11): 6312-8, 1998 Mar 13.
Article in English | MEDLINE | ID: mdl-9497359

ABSTRACT

Maclura pomifera agglutinin is a tetrameric plant seed lectin with high affinity for the tumor-associated T-antigen disaccharide, Galbeta1,3GalNAcalpha, and hence for many O-linked glycopeptide structures. Unlike members of most lectin families, it lacks both metal ions and Cys residues. The structure of its complex with Galbeta1,3GalNAc was determined to 2.2 by first using multiwavelength anomalous diffraction with a lead derivative of the native protein, and then using molecular replacement with the unrefined structure as a model to solve the structure of the complex. The subunits share the beta-prism architecture and three-fold pseudo-symmetry of the related lectin jacalin, with the 21-residue beta-chains in the center of the tetramer. Interactions with the GalNAc predominate in the binding of the disaccharide. It forms a network of H-bonds with only one side chain, from an Asp residue, the amino group of the N-terminal Gly of the alpha-chain, and peptide backbone atoms of two aromatic residues. The Gal moiety does not H-bond directly with residues in the same monomer, i.e. there is no true subsite for it, but there are interactions through two water molecules. In the crystal, it interacts with residues in the binding site of an adjacent tetramer. The minimum energy conformation expected for the disaccharide is retained, despite its mediating the tetramer-tetramer interactions in the crystal packing. The resulting lattice is comparable to those seen for complexes of other lectins with branched glycopeptides.


Subject(s)
Antigens, Tumor-Associated, Carbohydrate/chemistry , Biomarkers, Tumor/chemistry , Disaccharides/chemistry , Lectins/chemistry , Plant Lectins , Plant Proteins/chemistry , Carbohydrate Conformation , Crystallography/methods , Hydrogen Bonding , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation
15.
Acta Crystallogr D Biol Crystallogr ; 54(Pt 6 Pt 2): 1456-9, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-10089537

ABSTRACT

The histoblood-group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single-chain variable-domain antigen-binding fragment (scFv) gene based on the known sequence of an anti-blood-group-A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli. The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms. The corresponding variable-domain antigen-binding fragment (Fv) was generated by cleaving the VL-VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin-A-trisaccharide conjugate (KD = 290 microM). AC1001 Fv crystals grown in the presence of N-acetylgalactosamine diffracted to 0.93 A resolution. This is the first reported example of a crystal of an antibody antigen-binding fragment diffracting to atomic resolution.


Subject(s)
ABO Blood-Group System/immunology , Hemagglutinins/chemistry , Immunoglobulin Fragments/chemistry , Acetylgalactosamine/chemistry , Acetylgalactosamine/immunology , Animals , Antigen-Antibody Complex/chemistry , Carbohydrate Sequence , Cattle , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Hemagglutinins/genetics , Hemagglutinins/isolation & purification , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/isolation & purification , Molecular Sequence Data , Oligosaccharides/immunology , Oligosaccharides/metabolism , Oligosaccharides, Branched-Chain , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Serum Albumin, Bovine , Surface Plasmon Resonance
16.
J Biol Chem ; 272(9): 5533-8, 1997 Feb 28.
Article in English | MEDLINE | ID: mdl-9038159

ABSTRACT

The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in GM1) with tolerance for substitution with a second internal sialic acid (as in GD1b). Escherichia coli heat-labile enterotoxin bound to GM1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-GM1 and GD1b, respectively) but not substitution of the terminal galactose of GM1. Tetanus toxin showed a requirement for two internal sialic acid residues as in GD1b. Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.


Subject(s)
Cholera Toxin/metabolism , Peptide Fragments/metabolism , Receptors, Cell Surface/metabolism , Tetanus Toxin/metabolism , Biosensing Techniques , Carbohydrate Sequence , G(M1) Ganglioside/metabolism , Glycolipids/metabolism , Kinetics , Liposomes , Molecular Sequence Data , Neuromuscular Blocking Agents/metabolism , Salmonella , Surface Properties
17.
Indian J Pediatr ; 64(6): 747-53, 1997.
Article in English | MEDLINE | ID: mdl-10771916

ABSTRACT

The inflammatory diseases of external and middle ear are one of the commonest conditions encountered by the pediatric physician. Inner ear inflammations are less common and need special and urgent attention. Special management in each case requires detailed history, examination, necessary investigations and appropriate referral to otolaryngologist when necessary. The article is aimed to help formulate a plan in managing the inflammatory conditions of ear. Otalgia constitutes the most prominent of the symptoms in external and middle ear inflammations whereas vertigo, tinnitus and sensory hearing loss form the symptom complex for inner ear infections. It is necessary to understand the basic pathophysiology of the inflammatory condition to be able to institute a targetted treatment. The audiometry impedance studies, microbiology of discharge and occasionally ABR and CT scan from the mainstay of investigative workup. The treatment is specific and based on the precise diagnosis. It often requires the help of an otolaryngologist. Decisions may have to be made with regards to the need for any surgical intervention particularly in acute otitis media, an external canal abscess or an acute mastoiditis. A case of chronic otitis media with facial palsy or vertigo (labyrinthitis being a possibility) needs urgent intervention.


Subject(s)
Mastoiditis/diagnosis , Otitis Media with Effusion/diagnosis , Otitis Media, Suppurative/diagnosis , Child , Child, Preschool , Diagnosis, Differential , Female , Humans , Infant , Male , Mastoiditis/therapy , Otitis Media with Effusion/therapy , Otitis Media, Suppurative/therapy , Prognosis
18.
Indian J Pediatr ; 64(6): 755-61, 1997.
Article in English | MEDLINE | ID: mdl-10771917

ABSTRACT

Acute otitis media (AOM) and otitis media with effusion (OME) in children can present with a variety of middle ear effusions (MEE). Even though the character of the effusion may vary, the underlying pathogenesis is often similar. In the last decade, there has been an abundance of new information in the fields of immunobiology and immunochemistry to explain the chronicity of MEE. There are also studies examining the efficacy of vaccination and immunoprophylaxis for recurrent AOM. Diagnosis of otitis media (OM) in a child can be difficult but good visualization by pneumatic otoscopy improves the accuracy of diagnosis of OME. The development of increasing bacterial resistance to antimicrobial therapy reinforces the need to be more rational in treating AOM and OME. The variability of the natural history and the long term sequelae of OME makes medical management more difficult. All children with chronic MEE should have audiologic evaluation. Surgery is recommended should the condition be refractory to medical therapy or if the complications of MEE develop.


Subject(s)
Otitis Media with Effusion/diagnosis , Otitis Media with Effusion/therapy , Acute Disease , Anti-Bacterial Agents/administration & dosage , Child , Child, Preschool , Chronic Disease , Female , Humans , Infant , Male , Otologic Surgical Procedures/methods , Prognosis
19.
J Biol Chem ; 271(45): 28271-6, 1996 Nov 08.
Article in English | MEDLINE | ID: mdl-8910446

ABSTRACT

The genes encoding the alpha-2,3-sialyltransferases involved in lipooligosaccharide biosynthesis from Neisseria meningitidis and Neisseria gonorrhoeae have been cloned and expressed in Escherichia coli. A high sensitivity enzyme assay using a synthetic fluorescent glycosyltransferase acceptor and capillary electrophoresis was used to screen a genomic library of N. meningitidis MC58 L3 in a "divide and conquer" strategy. The gene, denoted lst, was found on a 2. 0-kilobase fragment of DNA, and its sequence was determined and then used to design probes to amplify and subsequently clone the corresponding lst genes from N. meningitidis 406Y L3, N. meningitidis M982B L7, and N. gonorrhoeae F62. Functional sialyltransferase was produced from the genes derived from both L3 N. meningitidis strains and the N. gonorrhoeae F62. However, the N. meningitidis M982B L7 gene contained a frameshift mutation that renders it inactive. The expression of the lst gene was easily detected using the enzyme assay, and the protein expression could be detected when an immunodetection tag was added to the COOH-terminal end of the protein. Using the synthetic acceptor N-acetyllactosamine-aminophenyl-(6-(5-(fluorescein-carboxamido)-hexan oic acid amide), the alpha-2,3 specificity of the enzyme was confirmed by NMR examination of the reaction product. The enzyme could also use synthetic acceptors with lactose or galactose as the saccharide portion. This study is the first example of the cloning, expression, and examination of alpha-2,3-sialyltransferase activity from a bacterial source.


Subject(s)
Antigens, Bacterial/biosynthesis , Lipopolysaccharides/biosynthesis , Neisseria gonorrhoeae/enzymology , Neisseria meningitidis/enzymology , Sialyltransferases/genetics , Amino Acid Sequence , Base Sequence , Blotting, Western , Cloning, Molecular , Gene Expression Regulation, Enzymologic , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Recombinant Proteins/chemistry , Sequence Alignment , Sequence Deletion , beta-Galactoside alpha-2,3-Sialyltransferase
20.
Glycoconj J ; 13(4): 575-83, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8872114

ABSTRACT

Electrospray mass spectrometry was used to identify precisely the proteolytic cleavage points within, and at the C-termini of, the proprotein forms of four Viciae lectins that give rise to their two-chain forms. The lectins examined were the pea and lentil lectins, favin and the Lathyrus odoratus lectin, which represent each of the four genera in this tribe. The molecular mass data showed single beta-chain forms for each lectin, with masses consistent with the available sequence and glycopeptide data, indicating that each came from a single proprotein. In contrast, the pea, lentil and L. odoratus alpha-chains occurred in as many as five forms, due to multiple C-terminal cleavage points. Only favin showed a single alpha-chain form. The alpha-chain mass data were again consistent with the sequence information available, except for the lentil lectin alpha-chain which was re-determined by protein sequencing. The two isolectin forms of this protein were shown to arise from alpha-chain species with and without residue Lys53. The mass spectrum of concanavalin A was also examined and both the single-chain form and the two fragment forms showed no evidence of C-terminal heterogeneity.


Subject(s)
Fabaceae/metabolism , Lectins/metabolism , Plants, Medicinal , Protein Processing, Post-Translational , Seeds/metabolism , Amino Acid Sequence , Lectins/chemistry , Mass Spectrometry , Molecular Sequence Data , Plant Lectins , Sequence Homology, Amino Acid
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