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Biomedical subjects

M J Adams

Publications and source records attributed to M J Adams.

At least 55 records · Page 3Linked to original sources
PubMed2000

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. RESULTS: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. CONCLUSIONS: The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

Amino Acid Sequence↗
PubMed2000

Sonographic appearance of nonoperatively treated Achilles tendon ruptures.

OBJECTIVE: This report describes the gray-scale and color Doppler sonographic appearance of healed Achilles tendon ruptures that have been treated nonoperatively. DESIGN AND PATIENTS: Eleven patients with acute Achilles tendon ruptures were studied with sonography in the acute setting and following nonoperative management of their torn tendon. RESULTS: On sonographic examination, healed tendons treated nonoperatively are characterized by mild residual distortion of the normal fibrillar architecture of the tendon, anterior bulging or irregularity of the healed tendon, and a hypoechoic area about the site of rupture. Less frequent observations include mildly increased color flow and calcification at the rupture site. The previously described findings of acute tears, including hematomas, gaps at the rupture site, hyperemic torn tendon ends, and markedly distorted fibrillar architecture, were seen to have resolved in this series. CONCLUSION: The Achilles tendon tear treated nonoperatively has a different sonographic appearance from that of a normal or acutely ruptured tendon.

Achilles Tendon↗
PubMed2000

Cardiac toxicity of intravenous terbutaline for the treatment of severe asthma in children: a prospective assessment.

OBJECTIVE: To examine the cardiac toxicity as measured by elevations in serum cardiac troponin T (cTnT) and to compare creatine kinase (CK) and creatine kinase MB (CK-MB) and findings on electrocardiography (ECG) as markers of cardiac toxicity with cTnT during the infusion of intravenous terbutaline for the treatment of severe asthma in children. STUDY DESIGN: Prospective cohort study of patients receiving intravenous terbutaline for severe asthma. RESULTS: Only 3 (10%) of the 29 patients had elevations in cTnT. Each underwent mechanical ventilation for >72 hours, which was the earliest point at which cTnT elevations were identified. Eighteen (62%) patients had an elevation in CK, and 3 had an elevation in CK-MB fraction without an elevated cTnT. Twenty (69%) patients had ECG findings consistent with ischemia, and 19 of these patients had the ischemic findings on their preterbutaline ECG. Elevations in CK and CK-MB and ischemic changes on ECG did not correlate with elevations in cTnT. Both mechanical ventilation (P =.02) and prolonged administration (>72 hours) of intravenous terbutaline (P =. 02) were significantly associated with elevations in cTnT. CONCLUSIONS: We found no clinically significant cardiac toxicity from the use of intravenous terbutaline for severe asthma as measured by serum cTnT elevations.

Adrenergic beta-Agonists↗
PubMed2000

A comparative study of functional assays for tissue factor pathway inhibitor using normal plasma and clinical samples.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type inhibitor that regulates the initiation of tissue factor-mediated coagulation. Recent reports in the literature have described variable results using different methodologies for TFPI measurement. In this study, we used one clotting and two amidolytic methodologies to assess TFPI functional levels. The study groups included normal healthy donors as well as patients with acute hepatitis, diabetes, coronary artery bypass graft operations, deep vein thrombosis, and prior to and during heparin therapy. The aims were to compare the results obtained in normal plasma using different assay systems, to compare TFPI levels in a range of clinical samples, including those previously not determined using a clotting methodology, and to report TFPI levels in patient groups previously not investigated. The results clearly demonstrate poor correlation between functional TFPI values using the different methodologies, highlighting the requirement for standardization.

Biological Assay↗
PubMed1999

Sequences of European wheat mosaic virus and oat golden stripe virus and genome analysis of the genus furovirus.

The complete nucleotide sequences of both RNAs of oat golden stripe virus (OGSV) and a wheat-infecting furovirus isolate from France, previously thought to be soil-borne wheat mosaic virus (SBWMV), have been determined. Both viruses had a similar genomic organisation to SBWMV and Chinese wheat mosaic virus, the two other furoviruses previously sequenced but had <70% nucleotides identical to them. The French isolate has been named European wheat mosaic virus (EWMV). Phylogenetic analyses supported the recognition of these isolates as distinct viruses in the genus Furovirus. Analysis of the coat protein readthrough domain on RNA2 of all furoviruses strongly predicts two mutually compatible conserved transmembrane domains that may be significant for fungus transmission. The second of these regions is eliminated by a deletion in the isolate of OGSV studied. Leaky opal (UGA) stop codons occur on both RNAs of all four furoviruses characterised and, in common with most other leaky opal codons identified in plant viruses, they are followed by a CGG codon.

Amino Acid Sequence↗
PubMed1999

6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme.

A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1. 44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4+/-1.4 microM and for NADP of 1.9+/-0.2 microM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2'-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.

Amino Acid Sequence↗
PubMed1999

Molecular analysis of barley yellow mosaic virus isolates from China.

The complete sequences of both RNAs of an isolate of barley yellow mosaic virus from Yancheng, Jiangsu province, China, were determined. The sequences resembled those of an isolate from Japan (96.8% identical nucleotides for RNA1; 95.7% for RNA2) more closely than one from Germany (93.9 and 91.0%, respectively). The greatest differences between the Chinese and Japanese isolates were in the 5'-UTRs of RNAs 1 and 2 (88.9 and 91.6% identical nucleotides, respectively) and there were also some other regions of difference in P1 (RNA2) and P3, CI, NIa and the 5' end of the coat protein (CP) (RNA1). Molecular differences between isolates from ten sites widely distributed in Eastern China were studied by sequencing RNA regions coding for the CP (RNA1) and the N-terminus of the P2 protein (RNA2). The P2 fragment was more variable than the CP, and phylogenetic analysis of both regions showed that Asian and European isolates formed distinct clusters. Differences between isolates were also revealed by single-strand conformation polymorphism of reverse transcription-polymerase chain reaction products, spanning the full lengths of both RNA1 and RNA2. However, molecular variations between isolates could not be linked to earlier results showing differences in cultivar response.

Base Sequence↗
PubMed1999

Complete sequence and genome properties of Chinese wheat mosaic virus, a new furovirus from China.

The complete nucleotide sequence of a virus infecting winter wheat in Shandong province, China has been determined. This was previously thought to be soil-borne wheat mosaic virus but, while the two viruses are related, they are only 75% (RNA1) and 63% (RNA2) identical at the nucleotide level, while the amino acid sequences share from 62% (19 kDa RNA2 product) to 84% (RNA1 replicase) identity. The analysis shows that the Chinese virus should be considered a new member of the genus Furovirus and has been named Chinese wheat mosaic virus (CWMV). A Cys-Gly ... Cys-Gly-X-X-His amino acid pattern was identified in the cysteine-rich protein of CWMV and those of several other plant virus genera, which seems likely to have some functional significance.

Amino Acid Sequence↗
PubMed1999

Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement.

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 A resolution data set and a dimer of the large beta + alpha domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 A. Crystals of the deletion mutant DeltaG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 A resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.

Amino Acid Sequence↗
PubMed1998

A 2.8 A resolution structure of 6-phosphogluconate dehydrogenase from the protozoan parasite Trypanosoma brucei: comparison with the sheep enzyme accounts for differences in activity with coenzyme and substrate analogues.

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 A, c=116.74 A, alpha=beta=90 degrees, gamma=120 degrees. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a beta-alpha-beta fold; while in most species the "fingerprint" sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.

Amino Acid Sequence↗
PubMed1998

On the mechanism of the reaction catalyzed by glucose 6-phosphate dehydrogenase.

The catalytic mechanism of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides was investigated by replacing three amino acids, His-240, Asp-177, and His 178, with asparagine, using site-directed mutagenesis. Each of the mutant enzymes was purified to homogeneity and characterized by substrate binding studies and steady-state kinetic analyses. The three-dimensional structure of the H240N glucose 6-phosphate dehydrogenase was determined at 2.5 A resolution. The results support a mechanism in which His-240 acts as the general base that abstracts the proton from the C1-hydroxyl group of glucose 6-phosphate, and the carboxylate group of Asp-177 stabilizes the positive charge that forms on His-240 in the transition state. The results also confirm the postulated role of His-178 in binding the phosphate moiety of glucose 6-phosphate.

Aspartic Acid↗
PubMed1998

Amino acid substitutions at the dimer interface of human glucose-6-phosphate dehydrogenase that increase thermostability and reduce the stabilising effect of NADP.

Over 100 mutations of the G6PD gene have been documented. With the construction of the molecular model of glucose-6-phosphate dehydrogenase, based on the structure of the bacterial Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, it has been possible to superimpose these amino acid changes on to the structure of the glucose-6-phosphate dehydrogenase molecule. There are a large number of severe disease causing mutations at the dimer interface which usually cause decreased thermostability. We have used this knowledge to predict amino acid changes which would effect an increase in the stability of the dimer. The aspartic acid at residue 421 was chosen as it is a negatively charged residue at the centre of the dimer interface in an area rich in negatively charged residues. This residue was changed to a neutrally charged alanine or asparagine, or a positively charged lysine or arginine. The thermostability of the enzyme was increased when residue 421 was neutral (A or N) and increased further when positive (K or R). NADP is known to exert a concentration dependent stabilising effect on the glucose-6-phosphate dehydrogenase dimer. However the concentration-dependent stabilising effect of NADP was reduced in the residue-421 substitutions in a manner which was inversely proportional to charge change. These results suggest that changes at the dimer interface can also affect the distant (> 20 A) NADP-binding site, and vice versa; an attempt has been made to explain these interactions based on the molecular model of human glucose-6-phosphate dehydrogenase.

Dimerization↗
PubMed1998

Characterization of the regulatory regions in the human desmoglein genes encoding the pemphigus foliaceous and pemphigus vulgaris antigens.

The adhesive proteins in the desmosome type of cell junction consist of two members of the cadherin superfamily, the desmogleins and desmocollins. Both desmogleins and desmocollins occur as at least three different isoforms with various patterns of expression. The molecular mechanisms controlling the differential expression of the desmosomal cadherin isoforms are not yet known. We have begun an investigation of desmoglein gene expression by cloning and analysing the promoters of the human genes coding for the type 1 and type 3 desmogleins (DSG1 and DSG3). The type 1 isoform is restricted to the suprabasal layers of the epidermis and is the autoantigen in the autoimmune blistering skin disease pemphigus foliaceous. The type 3 desmoglein isoform is also expressed in the epidermis, but in lower layers than the type 1 isoform, and is the autoantigen in pemphigus vulgaris. Phage lambda genomic clones were obtained containing 4.2 kb upstream of the translation start site of DSG1 and 517 bp upstream of the DSG3 start site. Sequencing of 660 bp upstream of DSG1 and 517 bp upstream of DSG3 revealed that there was no obvious TATA box, but a possible CAAT box was present at -238 in DSG1 and at -193 in DSG3 relative to the translation start site. Primer extension analysis and RNase protection experiments revealed four putative transcription initiation sites for DSG1 at positions -163, -151, -148 and -141, and seven closely linked sites for DSG3, the longest being at -140 relative to the translation start site. The sequences at these possible sites at -166 to -159 in DSG1 (TTCAGTCC) and at -124 to -117 in DSG3 (CTTAGACT) have some similarity to the initiator sequence (CTCANTCT) described for a TATA-less promoter often from -3 to +5, and the true transcription initiator site might therefore be the A residue in these sequences. There were two regions of similarity between the DSG1 and DSG3 promoters just upstream of the transcription initiation sites, of 20 and 13 bp, separated by 41 bp in DSG1 and 36 bp in DSG3. The significance of these regions of similarity remains to be elucidated, but the results suggest that they represent a point at which these two desmoglein genes are co-ordinately regulated. Analysis of the upstream sequences revealed GC-rich regions and consensus binding sites for transcription factors including AP-1 and AP-2. Exon boundaries were conserved compared with the classical cadherin E-cadherin, but the equivalent of the second cadherin intron was lacking. A 4.2 kb region of the human DSG1 promoter sequence was linked to the lacZ gene reporter gene in such a way that there was only one translation start site, and this construct was used to generate transgenic mice. We present the first transgenic analysis of a promoter region taken from a desmosomal cadherin gene. Our results suggest that the 4.2 kb upstream region of DSG1 does not contain all the regulatory elements necessary for correct expression of this gene but might have elements that regulate activity during hair growth.

Animals↗
PubMed1998

The effects of alcohol on coagulation and fibrinolytic factors: a controlled trial.

Light-to-moderate alcohol intake is associated with a reduced incidence of ischaemic cardiovascular events, whilst heavy alcohol intake can predispose individuals to stroke. Alcohol-induced changes in coagulation and fibrinolysis may be relevant and are the subject of this controlled trial of varying alcohol intake in 55 predominantly beer-drinking men. Following 4 weeks stabilization maintaining usual drinking habits, participants were randomized to either continue usual alcohol intake or to restrict alcohol by changing to low alcohol beer for 4 weeks. In a final 4 week period, they crossed over to low or usual alcohol intake, respectively. Comparing combined low and usual alcohol periods, an increase in mean weekly alcohol intake from 92 to 410 ml (mean daily intake from 13 to 58 ml) was associated with a decrease in plasma fibrinogen (by 11%, P < 0.001) and platelet count (3%, P < 0.05), but increases in factor VII (7%, P = 0.001), tissue plasminogen activator (tPA; 16%, P = 0.01) and plasminogen activator inhibitor-1 (PAI-1; 21%, P < 0.001). The ratio, tPA/PAI-1, fell from 0.50 to 0.44 (P = 0.02) confirming the relatively greater increase in PAI-1 with alcohol consumption. Two lipid-associated natural anticoagulants, tissue factor pathway inhibitor and beta 2-glycoprotein-I, did not change. The substantial reduction in plasma fibrinogen with alcohol intake may well contribute to the apparent protection alcohol confers against ischaemic coronary and cerebral events. The increase in factor VII and relatively greater increase in PAI-1 than tPA with alcohol intake may attenuate this benefit and indeed may sufficiently predispose individuals to thrombosis to contribute to the increased incidence of ischaemic stroke seen in heavier drinkers. The balance of anticoagulant and procoagulant and fibrinolytic effects in any individual may vary depending on quantity and type of alcoholic beverage ingested, as well as on genetic and other variables, all of which merit further study.

Adult↗
PubMed1997

Cloning and transcriptional analysis of the promoter of the human type 2 desmocollin gene (DSC2).

The desmocollins, together with the desmogleins, are members of the cadherin family and constitute the adhesive proteins of the desmosome type of cell-cell junction. Here we describe a study of the promoter of the human form of the DSC2 gene which is the equivalent of the first isoform expressed in the developing mouse embryo and that has the most widespread tissue distribution in epithelia and also in desmosome-bearing non-epithelial tissues. Analysis of the 5' upstream region by DNA sequencing and Southern blotting suggested that it contained a CpG island, and a major site of transcription initiation 201 bp upstream of the translation start site was found by RNase protection and primer extension. There were no obvious CCAAT or TATA boxes present. Analysis of 1.9 kb upstream of the translation start site revealed consensus binding sites for transcription factors including Ap-2 and Sp-1, and motifs common to the promoters of other epithelially expressed genes such as keratin 14 and the desmoglein genes DSG1 and DSG3. Deletion derivatives defined a promoter of 525 bp which was active in epithelial cells and in mouse blastocysts with an intact epithelium. This promoter showed reduced expression in non-epithelial cells.

3T3 Cells↗