Peter George - Academia.edu (original) (raw)
Papers by Peter George
The New Zealand medical journal
Homozygosity for the apolipoprotein (apo) E variant apoE2(158 Arg+Cys) invariably gives rise to d... more Homozygosity for the apolipoprotein (apo) E variant apoE2(158 Arg+Cys) invariably gives rise to dysbetalipopro- teinemia, and when associated with obesity or a gene for hyper- lipidemia, results in type I11 hyperlipoproteinemia. The associa- tion of the E212 phenotype with type IVIV hyperlipoproteinemia rather than type I11 hyperlipoproteinemia in identical twin brothers led us to investigate the primary structure of their
Nutrition Research, 2007
Coenzyme Q 10 (CoQ 10 ) is essential for every cell in the body, and deficiency has been implicat... more Coenzyme Q 10 (CoQ 10 ) is essential for every cell in the body, and deficiency has been implicated in many diseases. Studies to confirm the benefit of supplementation require efficacious supplementation. Previously we found Q-Gel to be highly bioavailable. The objective of the present study was to identify the most efficacious dose of Q-Gel for use in supplementation studies. In a randomized crossover design, 8 young healthy male volunteers received single doses of 60, 150, and 300 mg CoQ 10 via Q-Gel 30-mg capsules, and of 300 mg via 100-mg Q-Gel capsules. Doses were given after a 10-hour overnight fast, a week apart. Plasma was analyzed for CoQ 10 , total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triacylglycerols at baseline and at 2, 4, 6, 8, and 10 hours after supplement ingestion. Areas under the curve (AUCs) were calculated for each participant at each dose and compared by 1-way analysis of variance. AUC increased significantly between 60-and 150-mg doses (P b .001), but not between 150-and 300-mg doses (P = .198). A plateau in absorption occurs near 200 mg. AUC for the 300-mg dose via 100-mg capsules was significantly lower than that for 300 mg via 30-mg capsules (P b .001), which may be due to the lower ratio of CoQ 10 to oil in the 30-mg capsules or to the higher vitamin E content in the 100-mg capsules. We conclude that the most efficacious single dose of Q-Gel is 200 mg, and higher absorption is obtained using multiple smaller capsules.
Journal of Clinical Microbiology, 2003
Food Chemistry, 2003
... FJ de Zwart a , b , S. Slow Corresponding Author Contact Information , E-mail The Correspondi... more ... FJ de Zwart a , b , S. Slow Corresponding Author Contact Information , E-mail The Corresponding Author , a , RJ Payne a , M. Lever a , PM George a , JA Gerrard b and ST Chambers c. ... Cooking methods included boiling, steaming, microwaving, baking, and frying. ...
Journal of Biological Chemistry
Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human al-proteinase ... more Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human al-proteinase inhibitor, representing 10% of the soluble cell protein, has been synthesized in yeast. Two forms of this protein were isolated with one being analogous to the human plasma protein and the other having the amino acid valine replacing methionine at position 358 (the P1 position). Both proteins were more sensitive to heat inactivation than the plasma form, and both had shorter half-lives in rabbits. These differences were presumably due to the absence of carbohydrate. Each protein could bind neutrophil elastase at a rate only slightly slower than that of human plasma al-proteinase inhibitor. However, the valine variant was stable to oxidation, while the P1 methionine-containing protein was readily inactivated. The specificity of alproteinase inhibitor (methionine) was identical to that of the plasma form; however, the valine form could only effectively bind to neutrophil or pancreatic elastase, "trypsin-like" serine proteinases not being inactivated at all. These data indicate the potential importance of mutant forms of proteinase inhibitors, produced by recombinant DNA technology, as therapeutic agents for the inactivation of excess proteinases of a specific type in tissues.
The New Zealand medical journal
Hemoglobin
The recent report of the oxidation of beta 141 Leu in a New Zealand family with Hb Atlanta [ beta... more The recent report of the oxidation of beta 141 Leu in a New Zealand family with Hb Atlanta [ beta 75(E19)Leu-->Pro] prompted us to reinvestigate the original Hb Atlanta case from Georgia. Tryptic peptide maps showed that the modified beta CoT-14 peptide was present together with beta AT-14 in isopropanol precipitates of Hb Atlanta. Amino acid analysis confirmed that beta CoT-14 lacked leucine and mass spectrometry indicated that it had an increased mass of 16 Daltons. These findings support the proposition that the beta 75 Leu-->Pro substitution in the E helix is the direct cause of the beta 141 Leu oxidation.
British Journal of Haematology
Analyses of haemoglobin from a family with an unstable haemoglobin haemolytic anaemia demonstrate... more Analyses of haemoglobin from a family with an unstable haemoglobin haemolytic anaemia demonstrated that the affected individuals had three beta-globins, namely, normal (beta A), Atlanta (beta At) with a mutation of beta 75 Leu----Pro, and beta-Atlanta-Coventry (beta At-Co) with mutation of beta 75 Leu----Pro and beta 141 Leu deleted. These were present in the ratio 66:23:11 respectively. The structure of the beta-globin cluster, however, was found to be normal by Southern blotting; also cytogenetic analysis failed to show any abnormality. DNA sequence analyses demonstrated the presence of the beta At mutation in genomic DNA isolated from leucocytes but the Coventry deletion of 141 Leu in beta At-Co was not present in genomic DNA. PCR amplification of the beta-globin cDNA and direct sequencing of the product also failed to demonstrate the Coventry deletion. Thus, it appears that the absence of 141 Leu in the beta At-Co globin is a consequence of the beta At mutation in these patients and that both beta At and beta At-Co are the product of a single gene. This unusual conclusion is paralleled in the bizarre case of Hb Vicksburg where the deletion of a leucine at beta 75 is not coded for in genomic DNA.
Low plasma total cholesterol (TC) concentrations have been associated with higher mortality in ch... more Low plasma total cholesterol (TC) concentrations have been associated with higher mortality in chronic heart failure (CHF) (1). In the study of (1), lower serum TC was independently associated with total mortality in a CHF cohort, with increasing total serum cholesterol predicting survival (hazard ratio 0.64, 95% confidence interval 0.48 to 0.86), independent of the aetiology of CHF, age, left ventricular ejection fraction and exercise capacity.
The New Zealand medical journal
New Zealanders, because of a soil deficiency, have a low intake of selenium. To determine the imp... more New Zealanders, because of a soil deficiency, have a low intake of selenium. To determine the impact of this on the infant population in Christchurch. we have measured red cell and plasma selenium and the selenoenzyme, glutathione peroxidase, in 70 infants less than 12 months old and related these to age and diet. the infant population as a whole had mean plasma levels of selenium and glutathione peroxidase of 33 micrograms/L and 97 U/L compared with adult values of 74 micrograms/L and 150 U/L. Infant red cell levels of 0.30 mu g selenium and 9.0 U glutathione peroxidase per g haemoglobin were similar to those in adults. The selenium status of most breast fed infants after birth remained similar to that of cord blood. Mean plasma selenium and glutathione peroxidase levels in formula fed infants were about half those of breast fed infants, and their red cell selenium was also significantly lower. These did not increase until solids were introduced into the diet. The status of the infants reflected their diet, with the concentration of selenium in formulae being 3.9-5.2 micrograms/mL compared with a mean of 13.4 micrograms/mL in breast milk. since infants in more replete selenium areas show a gradual rise in blood selenium parameters after birth, this study suggests that formula fed and some breast fed infants in Christchurch receive an inadequate selenium intake. Consideration should be given to supplementing infant formulae and perhaps also the diet of pregnant and/or breast feeding mothers.
Thrombosis and Haemostasis
We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spe... more We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bbeta chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bbeta chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bbeta chains. This decrease of 48 Da in the intact protein is indicative of either a Bbeta 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bbeta 14 Arg --> Cys mutation was verified by PCR amplification and DNA sequence analysis.
Thrombosis and Haemostasis
Electrospray ionisation mass spectrometry was used to probe the structure of the new N-linked oli... more Electrospray ionisation mass spectrometry was used to probe the structure of the new N-linked oligosaccharide in fibrinogen Kaiserslautern (gamma 380 Lys-->Asn). The mass increase of 2177 Da in the new beta chain indicated the attachment of a fully sialylated biantennary oligosaccharide on the new Asn residue; the expected increase for this change being 2192 Da. Some 95% of the new oligosaccharide was in the disialylated state while only 5% of the endogenous gamma chain carbohydrate was disialylated in the control. Mass measurements of intact Kaiserslautern gamma chains after neuraminidase treatment of the native fibrinogen confirmed a total of three residues of sialic acid in the dominant isoform. Incubation with endoglycosidase F showed that the new oligosaccharide was more resistant to hydrolysis than the endogenous one. Recent X-ray analyses of covalently linked D domains show that position gamma 380 is distant from both the GPR binding pocket and the D-D interface. It appears that the polymerisation defect of this fibrinogen results from electrostatic repulsion between condensing protofibrils and that this is induced by the two new residues of sialic acid that are present on the new gamma chain.
Thrombosis and Haemostasis
We investigated the molecular basis of hypofibrinogenemia in a woman with a plasma fibrinogen of ... more We investigated the molecular basis of hypofibrinogenemia in a woman with a plasma fibrinogen of 1.0 mg/mL. After sequencing the coding region and intronic boundaries of all three fibrinogen genes a single heterozygous GAC-->TAC mutation was identified at codon 316 of the Bbeta gene. This Asp-->Tyr substitution segregated with the hypofibrinogenemia in the only other affected family member. Examination by SDS-PAGE, isoelectric focussing, reverse phase chromatography and electrospray ionisation (ESI) mass spectrometery, failed to detect expression of the new Bbeta chain in purified plasma fibrinogen. The absence of the variant chain was confirmed by ESI tryptic mapping; while the [M + 1 H] and [M + 2 H] ions of the affected peptide (MGPTELLIEMEDWK) were clearly visible at 1,692 and 847 m/z, there were no new signals (1,741 or 871 m/z) that would at indicate expression of the variant in plasma. Asp 316 and its gamma chain homologue (Asp 252) are conserved in all known species and this is the first report of a mutation at either of these. The residue appears to be critical in maintaining the structure of the five stranded sheet that forms the dominant structural feature of the D domains.
American Journal Of Pathology
The proposita suffered from liver cirrhosis and biopsy showed type 1 membrane-bound fiberglass in... more The proposita suffered from liver cirrhosis and biopsy showed type 1 membrane-bound fiberglass inclusions. The hepatic inclusion bodies were weakly periodic acid-Schiff diastase-positive, and on immunoperoxidase staining reacted specifically with antifibrinogen antisera. Coagulation investigations revealed low functional and antigenic fibrinogen together with a prolonged thrombin time of 37 seconds (normal, 17 to 22 seconds) suggestive of a hypodysfibrinogenemia. DNA sequencing of all three fibrinogen genes showed a single heterozygous mutation of GGG (Gly)3 CGG (Arg) at codon 284 of the ␥chain gene. However, examination of purified fibrinogen chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, and isoelectric focusing, failed to show any evidence of the mutant ␥ Br chain in plasma fibrinogen. This finding was substantiated by electrospray ionization mass spectrometry, which showed only a normal ␥ (and B) chain mass, but a large increase in the portion of their disialo isoforms. We speculate that misfolding of the variant protein causes hepatic retention and the subsequent hypofibrinogenemia, and that the functional defect (dysfibrinogenemia) results from hypersialylation of otherwise normal B and ␥ chains consequent to the liver cirrhosis. These conclusions were supported by studies on six other family members with hypofibrinogenemia, and essentially normal clotting times, who were heterozygous for the ␥284 Gly3 Arg mutation. Fibrinogen is synthesized in the liver as a 340-kd glycoprotein. The molecule is a dimer with each half consisting of three disulfide-linked polypeptide chains (A␣, B, and ␥) with nominal molecular weights of 66, 55, and 48 kd, respectively. 1,2 The symmetrical molecule consists of a central E domain joined to two peripheral D domains by triple-helix spacers. Single biantennary oligosaccharide side chains, of sequence, -NAcGlc 2 -Man-(Man-NAcGlc-Gal-NAcNeu) 2 , are attached to the B and ␥ chains at Asn 364 and 52, respectively. After activation by thrombin, the fibrin monomer spontaneously polymerizes through D:E interactions to form a half-staggered bimolecular array that is stabilized by noncovalent end-to-end D:D interactions between adjacent monomer units.
Thrombosis and Haemostasis
We investigated the molecular basis of hypofibrinogenaemia in a woman with a history of recurrent... more We investigated the molecular basis of hypofibrinogenaemia in a woman with a history of recurrent, pregnancy-associated bleeding, and miscarriage. She had a Clauss fibrinogen of 0.9 mg/ml and SDS PAGE of purified fibrinogen showed a normal pattern of chains. However careful inspection of reverse phase chain separation profiles showed apparent homozygosity for a more hydrophilic form of the gamma chain. DNA Sequencing showed only heterozygosity for a CGT-->GGT (Ala-->Gly) mutation at codon gamma82, but further sequencing showed an additional GT splice sequence mutation at the 5' end of intron 2 of the gamma gene. Translation of mRNA containing this intron would result in premature truncation explaining the phenotypic homozygosity of the gamma82 Ala-->Gly substitution. The patient's sister had a mild bleeding disorder with hypofibrinogenaemia and she too was a compound heterozygote for the y mutations. Her nephew had only the novel splice site mutation, while her mother and daughter inherited only the gamma82 Ala-->Gly substitution.
Blood
We investigated the molecular basis of hypofibrinogenemia in a man with a normal thrombin clottin... more We investigated the molecular basis of hypofibrinogenemia in a man with a normal thrombin clotting time. Protein analysis indicated equal plasma expression of 2 different Bbeta alleles, and DNA sequencing confirmed heterozygosity for a new Bbeta235 P-->L mutation. Protein analysis also revealed a novel gamma(D) chain, present at a ratio of 1:2 relative to the gamma(A) chain. Mass spectrometry indicated a 14 d decrease in the gamma(D)-chain mass, and DNA sequencing showed this was caused by a novel gamma82 A-->G substitution. DNA sequencing established heterozygosity for 2 further mutations: T-->C in intron 4 of the Aalpha gene and A-->C in the 3' noncoding region of the Bbeta gene. Studies on the man's daughter, together with plasma expression levels, discounted both the Aalpha and Bbeta mutations as the cause of the low fibrinogen, suggesting that the gamma82 mutation caused the hypofibrinogenemia. This was supported by analysis of 31 normal controls in whom the Bbeta mutations were found at polymorphic levels, with an allelic frequency of 5% for the Bbeta235 mutation and 42% for the Bbeta 3' untranslated mutation. The gamma82 mutation was, however, unique to the propositus. Residue gamma82 is located in the triple helix that separates the E and D domains, and aberrant packing of the helices may explain the decreased fibrinogen concentration. (Blood. 2000;95:1709-1713)
Clinical Chemistry
... Hayley J. Ridgway a , Stephen O. Brennan, Andrew P. Fellowes and Peter M. George ... L KCl, 1... more ... Hayley J. Ridgway a , Stephen O. Brennan, Andrew P. Fellowes and Peter M. George ... L KCl, 10 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L MgCl 2 , 200 µmol/L of each dNTP, 1 µmol/L of each primer, 1 µg of DNA template, and 2 units of Taq DNA polymerase (Boehringer Mannheim). ...
Postgraduate Medical Journal
A woman was screened for diabetes using glycated haemoglobin (HbA1c). Vastly different results we... more A woman was screened for diabetes using glycated haemoglobin (HbA1c). Vastly different results were obtained by high performance liquid chromatography (45%), immunoassay (2.9%), and affinity chromatography (4.2%) compared with the non-diabetic range of less than 6.4%. Mass spectral studies confirmed the presence a haemoglobin variant, haemoglobin Marseille-Long Island which had confounded interpretation by all methods.
Thrombosis and Haemostasis
The New Zealand medical journal
Homozygosity for the apolipoprotein (apo) E variant apoE2(158 Arg+Cys) invariably gives rise to d... more Homozygosity for the apolipoprotein (apo) E variant apoE2(158 Arg+Cys) invariably gives rise to dysbetalipopro- teinemia, and when associated with obesity or a gene for hyper- lipidemia, results in type I11 hyperlipoproteinemia. The associa- tion of the E212 phenotype with type IVIV hyperlipoproteinemia rather than type I11 hyperlipoproteinemia in identical twin brothers led us to investigate the primary structure of their
Nutrition Research, 2007
Coenzyme Q 10 (CoQ 10 ) is essential for every cell in the body, and deficiency has been implicat... more Coenzyme Q 10 (CoQ 10 ) is essential for every cell in the body, and deficiency has been implicated in many diseases. Studies to confirm the benefit of supplementation require efficacious supplementation. Previously we found Q-Gel to be highly bioavailable. The objective of the present study was to identify the most efficacious dose of Q-Gel for use in supplementation studies. In a randomized crossover design, 8 young healthy male volunteers received single doses of 60, 150, and 300 mg CoQ 10 via Q-Gel 30-mg capsules, and of 300 mg via 100-mg Q-Gel capsules. Doses were given after a 10-hour overnight fast, a week apart. Plasma was analyzed for CoQ 10 , total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triacylglycerols at baseline and at 2, 4, 6, 8, and 10 hours after supplement ingestion. Areas under the curve (AUCs) were calculated for each participant at each dose and compared by 1-way analysis of variance. AUC increased significantly between 60-and 150-mg doses (P b .001), but not between 150-and 300-mg doses (P = .198). A plateau in absorption occurs near 200 mg. AUC for the 300-mg dose via 100-mg capsules was significantly lower than that for 300 mg via 30-mg capsules (P b .001), which may be due to the lower ratio of CoQ 10 to oil in the 30-mg capsules or to the higher vitamin E content in the 100-mg capsules. We conclude that the most efficacious single dose of Q-Gel is 200 mg, and higher absorption is obtained using multiple smaller capsules.
Journal of Clinical Microbiology, 2003
Food Chemistry, 2003
... FJ de Zwart a , b , S. Slow Corresponding Author Contact Information , E-mail The Correspondi... more ... FJ de Zwart a , b , S. Slow Corresponding Author Contact Information , E-mail The Corresponding Author , a , RJ Payne a , M. Lever a , PM George a , JA Gerrard b and ST Chambers c. ... Cooking methods included boiling, steaming, microwaving, baking, and frying. ...
Journal of Biological Chemistry
Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human al-proteinase ... more Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human al-proteinase inhibitor, representing 10% of the soluble cell protein, has been synthesized in yeast. Two forms of this protein were isolated with one being analogous to the human plasma protein and the other having the amino acid valine replacing methionine at position 358 (the P1 position). Both proteins were more sensitive to heat inactivation than the plasma form, and both had shorter half-lives in rabbits. These differences were presumably due to the absence of carbohydrate. Each protein could bind neutrophil elastase at a rate only slightly slower than that of human plasma al-proteinase inhibitor. However, the valine variant was stable to oxidation, while the P1 methionine-containing protein was readily inactivated. The specificity of alproteinase inhibitor (methionine) was identical to that of the plasma form; however, the valine form could only effectively bind to neutrophil or pancreatic elastase, "trypsin-like" serine proteinases not being inactivated at all. These data indicate the potential importance of mutant forms of proteinase inhibitors, produced by recombinant DNA technology, as therapeutic agents for the inactivation of excess proteinases of a specific type in tissues.
The New Zealand medical journal
Hemoglobin
The recent report of the oxidation of beta 141 Leu in a New Zealand family with Hb Atlanta [ beta... more The recent report of the oxidation of beta 141 Leu in a New Zealand family with Hb Atlanta [ beta 75(E19)Leu-->Pro] prompted us to reinvestigate the original Hb Atlanta case from Georgia. Tryptic peptide maps showed that the modified beta CoT-14 peptide was present together with beta AT-14 in isopropanol precipitates of Hb Atlanta. Amino acid analysis confirmed that beta CoT-14 lacked leucine and mass spectrometry indicated that it had an increased mass of 16 Daltons. These findings support the proposition that the beta 75 Leu-->Pro substitution in the E helix is the direct cause of the beta 141 Leu oxidation.
British Journal of Haematology
Analyses of haemoglobin from a family with an unstable haemoglobin haemolytic anaemia demonstrate... more Analyses of haemoglobin from a family with an unstable haemoglobin haemolytic anaemia demonstrated that the affected individuals had three beta-globins, namely, normal (beta A), Atlanta (beta At) with a mutation of beta 75 Leu----Pro, and beta-Atlanta-Coventry (beta At-Co) with mutation of beta 75 Leu----Pro and beta 141 Leu deleted. These were present in the ratio 66:23:11 respectively. The structure of the beta-globin cluster, however, was found to be normal by Southern blotting; also cytogenetic analysis failed to show any abnormality. DNA sequence analyses demonstrated the presence of the beta At mutation in genomic DNA isolated from leucocytes but the Coventry deletion of 141 Leu in beta At-Co was not present in genomic DNA. PCR amplification of the beta-globin cDNA and direct sequencing of the product also failed to demonstrate the Coventry deletion. Thus, it appears that the absence of 141 Leu in the beta At-Co globin is a consequence of the beta At mutation in these patients and that both beta At and beta At-Co are the product of a single gene. This unusual conclusion is paralleled in the bizarre case of Hb Vicksburg where the deletion of a leucine at beta 75 is not coded for in genomic DNA.
Low plasma total cholesterol (TC) concentrations have been associated with higher mortality in ch... more Low plasma total cholesterol (TC) concentrations have been associated with higher mortality in chronic heart failure (CHF) (1). In the study of (1), lower serum TC was independently associated with total mortality in a CHF cohort, with increasing total serum cholesterol predicting survival (hazard ratio 0.64, 95% confidence interval 0.48 to 0.86), independent of the aetiology of CHF, age, left ventricular ejection fraction and exercise capacity.
The New Zealand medical journal
New Zealanders, because of a soil deficiency, have a low intake of selenium. To determine the imp... more New Zealanders, because of a soil deficiency, have a low intake of selenium. To determine the impact of this on the infant population in Christchurch. we have measured red cell and plasma selenium and the selenoenzyme, glutathione peroxidase, in 70 infants less than 12 months old and related these to age and diet. the infant population as a whole had mean plasma levels of selenium and glutathione peroxidase of 33 micrograms/L and 97 U/L compared with adult values of 74 micrograms/L and 150 U/L. Infant red cell levels of 0.30 mu g selenium and 9.0 U glutathione peroxidase per g haemoglobin were similar to those in adults. The selenium status of most breast fed infants after birth remained similar to that of cord blood. Mean plasma selenium and glutathione peroxidase levels in formula fed infants were about half those of breast fed infants, and their red cell selenium was also significantly lower. These did not increase until solids were introduced into the diet. The status of the infants reflected their diet, with the concentration of selenium in formulae being 3.9-5.2 micrograms/mL compared with a mean of 13.4 micrograms/mL in breast milk. since infants in more replete selenium areas show a gradual rise in blood selenium parameters after birth, this study suggests that formula fed and some breast fed infants in Christchurch receive an inadequate selenium intake. Consideration should be given to supplementing infant formulae and perhaps also the diet of pregnant and/or breast feeding mothers.
Thrombosis and Haemostasis
We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spe... more We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bbeta chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bbeta chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bbeta chains. This decrease of 48 Da in the intact protein is indicative of either a Bbeta 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bbeta 14 Arg --> Cys mutation was verified by PCR amplification and DNA sequence analysis.
Thrombosis and Haemostasis
Electrospray ionisation mass spectrometry was used to probe the structure of the new N-linked oli... more Electrospray ionisation mass spectrometry was used to probe the structure of the new N-linked oligosaccharide in fibrinogen Kaiserslautern (gamma 380 Lys-->Asn). The mass increase of 2177 Da in the new beta chain indicated the attachment of a fully sialylated biantennary oligosaccharide on the new Asn residue; the expected increase for this change being 2192 Da. Some 95% of the new oligosaccharide was in the disialylated state while only 5% of the endogenous gamma chain carbohydrate was disialylated in the control. Mass measurements of intact Kaiserslautern gamma chains after neuraminidase treatment of the native fibrinogen confirmed a total of three residues of sialic acid in the dominant isoform. Incubation with endoglycosidase F showed that the new oligosaccharide was more resistant to hydrolysis than the endogenous one. Recent X-ray analyses of covalently linked D domains show that position gamma 380 is distant from both the GPR binding pocket and the D-D interface. It appears that the polymerisation defect of this fibrinogen results from electrostatic repulsion between condensing protofibrils and that this is induced by the two new residues of sialic acid that are present on the new gamma chain.
Thrombosis and Haemostasis
We investigated the molecular basis of hypofibrinogenemia in a woman with a plasma fibrinogen of ... more We investigated the molecular basis of hypofibrinogenemia in a woman with a plasma fibrinogen of 1.0 mg/mL. After sequencing the coding region and intronic boundaries of all three fibrinogen genes a single heterozygous GAC-->TAC mutation was identified at codon 316 of the Bbeta gene. This Asp-->Tyr substitution segregated with the hypofibrinogenemia in the only other affected family member. Examination by SDS-PAGE, isoelectric focussing, reverse phase chromatography and electrospray ionisation (ESI) mass spectrometery, failed to detect expression of the new Bbeta chain in purified plasma fibrinogen. The absence of the variant chain was confirmed by ESI tryptic mapping; while the [M + 1 H] and [M + 2 H] ions of the affected peptide (MGPTELLIEMEDWK) were clearly visible at 1,692 and 847 m/z, there were no new signals (1,741 or 871 m/z) that would at indicate expression of the variant in plasma. Asp 316 and its gamma chain homologue (Asp 252) are conserved in all known species and this is the first report of a mutation at either of these. The residue appears to be critical in maintaining the structure of the five stranded sheet that forms the dominant structural feature of the D domains.
American Journal Of Pathology
The proposita suffered from liver cirrhosis and biopsy showed type 1 membrane-bound fiberglass in... more The proposita suffered from liver cirrhosis and biopsy showed type 1 membrane-bound fiberglass inclusions. The hepatic inclusion bodies were weakly periodic acid-Schiff diastase-positive, and on immunoperoxidase staining reacted specifically with antifibrinogen antisera. Coagulation investigations revealed low functional and antigenic fibrinogen together with a prolonged thrombin time of 37 seconds (normal, 17 to 22 seconds) suggestive of a hypodysfibrinogenemia. DNA sequencing of all three fibrinogen genes showed a single heterozygous mutation of GGG (Gly)3 CGG (Arg) at codon 284 of the ␥chain gene. However, examination of purified fibrinogen chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, and isoelectric focusing, failed to show any evidence of the mutant ␥ Br chain in plasma fibrinogen. This finding was substantiated by electrospray ionization mass spectrometry, which showed only a normal ␥ (and B) chain mass, but a large increase in the portion of their disialo isoforms. We speculate that misfolding of the variant protein causes hepatic retention and the subsequent hypofibrinogenemia, and that the functional defect (dysfibrinogenemia) results from hypersialylation of otherwise normal B and ␥ chains consequent to the liver cirrhosis. These conclusions were supported by studies on six other family members with hypofibrinogenemia, and essentially normal clotting times, who were heterozygous for the ␥284 Gly3 Arg mutation. Fibrinogen is synthesized in the liver as a 340-kd glycoprotein. The molecule is a dimer with each half consisting of three disulfide-linked polypeptide chains (A␣, B, and ␥) with nominal molecular weights of 66, 55, and 48 kd, respectively. 1,2 The symmetrical molecule consists of a central E domain joined to two peripheral D domains by triple-helix spacers. Single biantennary oligosaccharide side chains, of sequence, -NAcGlc 2 -Man-(Man-NAcGlc-Gal-NAcNeu) 2 , are attached to the B and ␥ chains at Asn 364 and 52, respectively. After activation by thrombin, the fibrin monomer spontaneously polymerizes through D:E interactions to form a half-staggered bimolecular array that is stabilized by noncovalent end-to-end D:D interactions between adjacent monomer units.
Thrombosis and Haemostasis
We investigated the molecular basis of hypofibrinogenaemia in a woman with a history of recurrent... more We investigated the molecular basis of hypofibrinogenaemia in a woman with a history of recurrent, pregnancy-associated bleeding, and miscarriage. She had a Clauss fibrinogen of 0.9 mg/ml and SDS PAGE of purified fibrinogen showed a normal pattern of chains. However careful inspection of reverse phase chain separation profiles showed apparent homozygosity for a more hydrophilic form of the gamma chain. DNA Sequencing showed only heterozygosity for a CGT-->GGT (Ala-->Gly) mutation at codon gamma82, but further sequencing showed an additional GT splice sequence mutation at the 5' end of intron 2 of the gamma gene. Translation of mRNA containing this intron would result in premature truncation explaining the phenotypic homozygosity of the gamma82 Ala-->Gly substitution. The patient's sister had a mild bleeding disorder with hypofibrinogenaemia and she too was a compound heterozygote for the y mutations. Her nephew had only the novel splice site mutation, while her mother and daughter inherited only the gamma82 Ala-->Gly substitution.
Blood
We investigated the molecular basis of hypofibrinogenemia in a man with a normal thrombin clottin... more We investigated the molecular basis of hypofibrinogenemia in a man with a normal thrombin clotting time. Protein analysis indicated equal plasma expression of 2 different Bbeta alleles, and DNA sequencing confirmed heterozygosity for a new Bbeta235 P-->L mutation. Protein analysis also revealed a novel gamma(D) chain, present at a ratio of 1:2 relative to the gamma(A) chain. Mass spectrometry indicated a 14 d decrease in the gamma(D)-chain mass, and DNA sequencing showed this was caused by a novel gamma82 A-->G substitution. DNA sequencing established heterozygosity for 2 further mutations: T-->C in intron 4 of the Aalpha gene and A-->C in the 3' noncoding region of the Bbeta gene. Studies on the man's daughter, together with plasma expression levels, discounted both the Aalpha and Bbeta mutations as the cause of the low fibrinogen, suggesting that the gamma82 mutation caused the hypofibrinogenemia. This was supported by analysis of 31 normal controls in whom the Bbeta mutations were found at polymorphic levels, with an allelic frequency of 5% for the Bbeta235 mutation and 42% for the Bbeta 3' untranslated mutation. The gamma82 mutation was, however, unique to the propositus. Residue gamma82 is located in the triple helix that separates the E and D domains, and aberrant packing of the helices may explain the decreased fibrinogen concentration. (Blood. 2000;95:1709-1713)
Clinical Chemistry
... Hayley J. Ridgway a , Stephen O. Brennan, Andrew P. Fellowes and Peter M. George ... L KCl, 1... more ... Hayley J. Ridgway a , Stephen O. Brennan, Andrew P. Fellowes and Peter M. George ... L KCl, 10 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L MgCl 2 , 200 µmol/L of each dNTP, 1 µmol/L of each primer, 1 µg of DNA template, and 2 units of Taq DNA polymerase (Boehringer Mannheim). ...
Postgraduate Medical Journal
A woman was screened for diabetes using glycated haemoglobin (HbA1c). Vastly different results we... more A woman was screened for diabetes using glycated haemoglobin (HbA1c). Vastly different results were obtained by high performance liquid chromatography (45%), immunoassay (2.9%), and affinity chromatography (4.2%) compared with the non-diabetic range of less than 6.4%. Mass spectral studies confirmed the presence a haemoglobin variant, haemoglobin Marseille-Long Island which had confounded interpretation by all methods.
Thrombosis and Haemostasis