Further research for consideration in 'the A2 milk case'.
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Animals↗
Biomedical subjects
A J Clarke
Publications and source records attributed to A J Clarke.
Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients.
MECP2 mutations are identifiable in approximately 80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.
Adolescent↗
ALK positive inflammatory myofibroblastic tumour of the pineal region.
Inflammatory myofibroblastic tumours (IMTs) are an uncommon spindle cell neoplasm with a dense inflammatory infiltrate, usually encountered in children. IMTs of the central nervous system are extremely rare. This report describes the case of an IMT in a 61 year old man, in the pineal region. The tumour was completely excised, and immunohistochemistry demonstrated anaplastic lymphoma kinase 1 expression. There was no tumour recurrence during 18 months of follow-up. Our case extends both the age range and sites of occurrence of this rare tumour.
Anaplastic Lymphoma Kinase↗
Inhibition of membrane-bound lytic transglycosylase B by NAG-thiazoline.
The lytic transglycosylases cleave the bacterial cell wall heteropolymer peptidoglycan with the same specificity as the muramidases (lysozymes), between the N-acetylmuramic acid and N-acetylglucosamine residues, with the concomitant formation of a 1,6-anhydromuramoyl residue. The putative catalytic residue in the family 3 lytic transglycosylase from Pseudomonas aeruginosa, Glu162 as identified by sequence alignment to the homologous enzyme from Escherichia coli, was replaced with both Ala and Asp by site-directed mutagenesis. Neither mutant enzyme differed structurally from the wild-type enzyme, as judged by CD spectroscopy, but both were enzymatically inactive confirming the essential role of Glu162 in the mechanism of action of this lytic transglycosylase. The beta-hexosaminidase inhibitor NAG-thiazoline was shown to inhibit the activity of lytic transglycosylase activity, thus providing the first direct evidence that the formation of the 1,6-anhydromuramoyl residue may proceed through an oxazolinium ion intermediate involving anchimeric assistance. Using surface plasmon resonance and difference absorbance spectroscopy, Kd values of 1.8 and 1.4 mM, respectively, were determined for NAG thiazoline, while its parent compound N-acetylglucosamine neither inhibited nor appeared to bind the lytic transglycosylase with any significant affinity.
Acetylglucosamine↗
Newborn screening for Duchenne muscular dystrophy.
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Female↗
Implications of carrier identification in newborn screening for cystic fibrosis.
OBJECTIVE: To investigate the psychosocial implications for families whose infant was identified as a cystic fibrosis carrier by newborn screening. DESIGN: Prospective psychosocial assessment. SETTING: Primary care. RESPONDENTS STUDY: (a) families of an affected infant identified by screening (n = 9); (b) families of a carrier infant identified by screening (n = 10). CONTROL: group of mothers from the general population (n = 82). INTERVENTIONS: Questionnaires and semistructured interviews. MAIN OUTCOME MEASURES: Attitude to screening, assessments of the mother/baby relationship, anxiety, wellbeing. RESULTS: All families were in favour of screening, with no evidence that the mother/baby relationship, anxiety or wellbeing had been adversely affected. Parents, however, did identify problems in terms of the service delivery protocol and genetic counselling practice. CONCLUSION: Six months after disclosure, carrier identification was not perceived by parents to be problematic.
Anxiety↗
Newborn screening for Duchenne muscular dystrophy: a psychosocial study.
OBJECTIVE: To evaluate the psychosocial implications of newborn screening for Duchenne muscular dystrophy. DESIGN: Prospective psychosocial assessment. SETTING: Primary care. RESPONDENTS STUDY: (a) families of an affected boy identified by screening (n = 20); (b) families of a boy with a transient screening abnormality (n = 18). CONTROL: (a) families of a boy with a later clinical diagnosis (n = 16); (b) random sample of mothers of boys aged 6-9 months (n = 43). INTERVENTIONS: Questionnaires and semistructured interviews. MAIN OUTCOME MEASURES: Attitudes to newborn screening and impact of screening on mother-baby relationship, anxiety/wellbeing, and reproductive patterning within families of an affected boy. RESULTS: Most families of an affected boy were in favour of newborn screening on the grounds of reproductive choice and time to prepare emotionally and practically. There was no evidence of any long term disruption to the mother-baby relationship. Anxiety levels for the screened group were slightly above threshold but returned to normal during the period of the study. There was no evidence, from anxiety or wellbeing scores, that the transient group had suffered any disadvantage. Although the profile of the screened and later clinically diagnosed cohorts was similar after diagnosis, when boys from the screened cohort were 4 years old and more socially aware, their profile was more positive. There was evidence that reproductive patterning had been modified, and four fetuses carrying a mutation causing Duchenne muscular dystrophy were terminated. CONCLUSION: A case can be made for newborn screening provided that the test is optional, a rigorous protocol for service delivery is used, and an infrastructure providing continuing support is in place.
Anxiety↗
Hyponatraemia without hypovolaemia.
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Aged↗
Differentiation of bacterial autolysins by zymogram analysis.
The use of zymograms in which the bacterial cell wall heteropolymer peptidoglycan is incorporated into the resolving gel of SDS-PAGE has led to the identification of various SDS stable peptidoglycan hydrolases (autolysins). To examine the specificity of autolysins with respect to O-acetylated peptidoglycan, a discontinuous SDS-PAGE system has been developed that operates under neutral conditions. [Bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (Bis-Tris) buffers are employed with pH 6.8 and 6.3 for the separating and stacking gels, respectively, while the anode buffer N-2-acetamido-2-hydroxyethanesulfonic acid (Aces)-HCl and the Bis-Tris cathode buffer both had a pH of 6.8. These conditions resulted in a relative trailing ion mobility of 0.349 and 0.137 in the resolving and staking gel, respectively, under room temperature conditions. Peptides and proteins were resolved in the 3-100 kDa range with a 10% acrylamide resolving gel. Comparison of zymograms that incorporated unacetylated or chemically O-acetylated peptidoglycan revealed the specificity of hen egg-white lysozyme for the unacetylated material. A preliminary analysis of the autolysins produced by the urinary tract pathogen Proteus mirabilis indicated that some enzymes were specific for either O-acetylated or non-O-acetylated peptidoglycan while others displayed no clear preference toward either of the two substrates.
Acetylation↗
Identification and characterization of a bovine myosin light chain-1 fast polymorphism.
This study aimed to identify genes or gene products associated with high lean muscle mass in bovines that may serve as potential markers for selection. An animal with a genetic predisposition to high lean muscle mass, the Belgium Blue, was chosen as a model to compare with the Holstein Friesian, a model that does not. Two-dimensional polyacrylamide gel electrophoresis analysis was utilized to compare the exhibited skeletal muscle proteome between the two animal types at two stages of foetal development. A previously uncharacterized polymorphism of a high expression myofibrillar protein, myosin light chain 1 fast (MLC-1f), was observed. The characterization of this polymorphism revealed a two amino acid insertion in a part of the protein that has been implicated in modulating myosin S1 ATPase activity. Furthermore, this polymorphism was shown to be the product of two alleles that are codominant. Screening studies were carried out on selected herds and showed a very high frequency of one allele. Both isoforms of MLC-1f were produced by recombinant means and purified. The recombinant proteins were exchanged into purified myosin hexamers that were then subject to assays measuring ATP consumption. The sensitivity of the assay utilized could not reveal any significant difference in ATPase activity between hexamers containing one or the other isoform.
Actins↗
Identification of four families of peptidoglycan lytic transglycosylases.
The lytic transglycosylases are a class of autolysins which cleave the bacterial cell wall heteropolymer peptidoglycan (murein) to facilitate its biosynthesis and turnover. A search of the National Center for Biotechnology Information (NCBI) databases using the primary sequences of the six characterized lytic transglycosylases of Escherichia coli, a membrane-bound form of the enzyme from Pseudomonas aeruginosa, and the endolysins of lambda bacteriophage permitted the identification of a total of 127 known and hypothetical enzymes from a wide variety of bacteria and bacteriophage. These amino acid sequences have been arranged into four families based on alignments, and consensus motifs have been identified. Family 1 represents a superfamily comprising 86 sequences which are subdivided into five (1A--1E) subfamilies.
Amino Acid Motifs↗
Three-component-mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3.
Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.
Acetyltransferases↗
Overexpression and characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.
The gene coding for aminoglycoside 2'-N-acetyltransferase Ia [AAC(2')-Ia] from Providencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.
Acetyltransferases↗
Identification of residues involved in catalytic activity of the inverting glycosyl transferase WbbE from Salmonella enterica serovar borreze.
Synthesis of the O:54 O antigen of Salmonella enterica is initiated by the nonprocessive glycosyl transferase WbbE, assigned to family 2 of the glycosyl transferase enzymes (GT2). GT2 enzymes possess a characteristic N-terminal domain, domain A. Based on structural data from the GT2 representative SpsA (S. J. Charnock and G. J. Davies, Biochemistry 38:6380-6385, 1999), this domain is responsible for nucleotide binding. It possesses two invariant Asp residues, the first forming a hydrogen bond to uracil and the second coordinating a Mn(2+) ion. Site-directed replacement of Asp41 (D41A) of WbbE, the analogue of the first Asp residue of SpsA, revealed that this is not required for activity. WbbE possesses three Asp residues near the position analogous to the second conserved residue. Whereas D95A reduced WbbE activity, activity in D93A and D96A mutants was abrogated, suggesting that either D93 or D96 may coordinate the Mn(2+) ion. Our studies also identified a C-terminal region of sequence conservation in 22 GT2 members, including WbbE. SpsA was not among these. This region is characterized by an ED(Y) motif. The Glu and Asp residues of this motif were individually replaced in WbbE. E180D in WbbE had greatly reduced activity, and an E180Q replacement completely abrogated activity; however, D181E had no effect. E180 is predicted to reside on a turn. Combined with the alignment of the motif with potential catalytic residues in the GT2 enzymes ExoM and SpsA, we speculate that E180 is the catalytic residue of WbbE. Sequence and predicted structural divergence in the catalytic region of GT2 members suggests that this is not a homogeneous family.
Amino Acid Motifs↗
Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae.
D-Galactan I is an O-antigenic polymer with the repeat unit structure [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of D-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of D-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The D-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into D-galactan I biosynthesis by the bifunctional wbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of D-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the D-galactan I polymer requires WbbM for Galp transferase, together with Galf transferase activity provided by WbbO. Comparison of the biosynthetic pathways for D-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.
Bacterial Proteins↗
Assay for lytic transglycosylases: a family of peptidoglycan lyases.
An assay has been developed to monitor the activity of the lytic transglycosylases which does not involve the use of radiolabel. Samples of lytic transglycosylase were incubated with isolated and purified insoluble peptidoglycan as substrate for varying lengths of time. Residual insoluble material was removed by ultracentrifugation in a microfuge and the solubilized components were treated with sodium borohydride prior to acid hydrolysis. The optimal conditions for this acid hydrolysis were established to be incubation at 96 degrees C for 1 h in 6 M HCl, in vacuo. The hydrolyzed samples were subjected to amino acid/sugar analysis by cation-exchange chromatography on a Beckman System Gold amino acid analyzer. To effect a clear resolution of muramic acid from serine and glutamic acid, the equilibration buffer was modified to be composed of 33 mM sodium citrate, pH 3.12. The product of the lyase reaction of the lytic transglycosylases are 1,6-anhydromuramyl residues, which are not reduced by the sodium borohydride treatment. On the other hand, the muramyl residues arising at the reducing ends of peptidoglycan after treatment with muramidases (hydrolyases) are reduced to muramitol residues, which elute from the amino acid analyzer prior to aspartic acid. This assay thus distinguishes the activity of the two enzymes and was applied to determine the initial activities of increasing concentrations of a soluble derivative of lytic transglycosylase B from the opportunistic pathogen Pseudomonas aeruginosa.
Amino Acids↗
The role of tryptophan residues in substrate binding to catalytic domains A and B of xylanase C from Fibrobacter succinogenes S85.
Oxidation of the isolated catalytic domain B of xylanase C (XynC-B) from Fibrobacter succinogenes with N-bromosuccinimide (NBS) resulted in the modification of five of the seven Trp residues present in the enzyme. Hydrolytic activity of the enzyme was rapidly lost upon initiation of oxidation as a molar ratio of about two NBS molecules per molar equivalent of protein was sufficient to cause 50% inhibition of enzyme activity, and the addition of five molar equivalents of NBS resulted in less than 10% activity. Pre-incubation of XynC-B with the competitive inhibitor D-xylose resulted in the apparent protection of two Trp residues from oxidation. Xylose protection of the enzyme also resulted in a maintenance of activity, with 60% activity still evident after addition of 8-9 molar equivalents of NBS. This protection from inactivation was enhanced by the inclusion of xylohexaose in reaction mixtures. Under these conditions, however, a further Trp residue was protected from NBS oxidation. The three protected Trp residues were identified as Trp135, Trp161 and Trp202 by differential labelling and peptide mapping of NBS-oxidized preparations of the xylanase employing a combination of electrospray mass spectroscopic analysis and N-terminal sequencing. By analogy to the known structures of the family 11 xylanases, the fully conserved Trp202 residue is located on the only alpha-helix present in the enzymes, at the interface between it and the back of the beta-sheet which forms the active site cleft. Trp135 represents a highly conserved aromatic residue in family 11, but it is replaced with Thr in domain A of F. succinogenes xylanase C. To investigate the role of Trp135 in conferring the different activity profile of domain B relative to domain A, the Trp135Thr and Trp135Ala derivatives of domain B were prepared by site-directed mutagenesis. However, the kinetic parameters of the two domain B derivatives were not significantly different compared to the wild-type enzyme as reflected by K(M) and k(cat) values and product distribution profiles. Similar results were obtained with the Trp161Ala derivative of domain B, indicating that these two residues do not directly participate in the binding of substrate but likely form the foundation for binding subsite 2.
Amino Acid Sequence↗
Characterization of function and activity of domains A, B and C of xylanase C from Fibrobacter succinogenes S85.
Xylanase C from the ruminant bacterium Fibrobacter succinogenes is comprised of two catalytic domains, A and B, and a third domain, C, of unknown function. The DNA coding for domains A and B of xylanase C were separately cloned and expressed in Escherichia coli as fusion proteins with glutathione-S:-transferase. The fusion proteins were isolated by affinity chromatography on glutathione-Sepharose 4B, cleaved with thrombin and the released xylanase C catalytic domains A and B were purified to apparent homogeneity by anion-exchange chromatography on Mono Q. Electrospray mass spectrometry provided a molecular mass of 27 818 Da (expected, 27 820 Da) for domain B. The pH and temperature optima for activity of domain B on oat spelt xylan were 5.0 and 52 degrees C, respectively. A kinetic analysis of the activity of the catalytic domain A on oat spelt xylan, birch wood xylan and xylooligomers at pH 6.5 and 37 degrees C provided data significantly different to those obtained previously with a protease-derived form of the enzyme [Zhu et al. (1994) J. Bacteriol. 176, 3885-3894]. The isolated domain A was more active on barley-glucan than the protease-derived form and its affinity for birch wood xylan was enhanced resulting in greater overall catalytic efficiency as reflected by k(cat)/K:(M) values. Likewise, significant differences in the Michaelis-Menten parameters K:(M), k(cat) and k(cat)/K:(M) were obtained with domain B compared with values previously reported with this domain attached to domain C. In general, the presence of domain C appeared to decrease the overall efficiency of domain B 7- and 36-fold with birch wood xylan and xylopentaose as substrates, respectively, as reflected by values of k(cat)/K:(M). The removal of domain C also affected the mode of action of domain B such that it more closely resembled that of catalytic domain A. However, no change in either pH and temperature optima or stability were found with domain B compared with the combined domains B and C. The function of domain C remains unknown, but hydrophobic cluster analysis indicated that it may belong to a class of dockerin domains involved in the protein-protein interactions of cellulolytic and xylanolytic complexes.
Amino Acid Sequence↗