Biomedical subjects

L J Bruce

Publications and source records attributed to L J Bruce.

At least 19 recordsLinked to original sources

PubMed2000

Band 3 mutations, renal tubular acidosis and South-East Asian ovalocytosis in Malaysia and Papua New Guinea: loss of up to 95% band 3 transport in red cells.

We describe three mutations of the red-cell anion exchangerband 3 (AE1, SLC4A1) gene associated with distalrenal tubular acidosis (dRTA) in families from Malaysia and Papua NewGuinea: Gly(701)-->Asp (G701D), Ala(858)-->Asp(A858D) and deletion of Val(850) (DeltaV850). The mutationsA858D and DeltaV850 are novel; all three mutations seem to berestricted to South-East Asian populations. South-East Asianovalocytosis (SAO), resulting from the band 3 deletion of residues400-408, occurred in many of the families but did not itselfresult in dRTA. Compound heterozygotes of each of the dRTA mutationswith SAO all had dRTA, evidence of haemolytic anaemia and abnormal red-cell properties. The A858D mutation showed dominant inheritance and therecessive DeltaV850 and G701D mutations showed a pseudo-dominantphenotype when the transport-inactive SAO allele was also present. Red-cell and Xenopus oocyte expression studies showed that theDeltaV850 and A858D mutant proteins have greatly decreased aniontransport when present as compound heterozygotes (DeltaV850/A858D,DeltaV850/SAO or A858D/SAO). Red cells with A858D/SAO had only 3% ofthe SO(4)(2-) efflux of normal cells, thelowest anion transport activity so far reported for human red cells. The results suggest dRTA might arise by a different mechanism for eachmutation. We confirm that the G701D mutant protein has an absoluterequirement for glycophorin A for movement to the cell surface. Wesuggest that the dominant A858D mutant protein is possibly mis-targetedto an inappropriate plasma membrane domain in the renal tubular cell,and that the recessive DeltaV850 mutation might give dRTA because ofits decreased anion transport activity.

Acidosis, Renal Tubular↗

PubMed2000

The low-frequency MNS blood group antigens Ny(a) (MNS18) and Os(a) (MNS38) are associated with GPA amino acid substitutions.

BACKGROUND: Antigens of the MNS blood group system are located on two sialoglycoproteins, GPA and GPB, encoded by GYPA and GYPB. The molecular backgrounds of the low-frequency antigens Ny(a) and Os(a) are not known. STUDY DESIGN AND METHODS: Immunoblotting and a monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay were used to analyze Os(a). PCR-amplified products of the coding exons of GYPA were studied by single-strand conformation polymorphism analysis, and exon 3 was sequenced. Synthetic peptides were used in hemagglutination-inhibition tests. RESULTS: Sequencing of GYPA exon 3 of two unrelated Ny(a+) persons revealed heterozygosity for a T194A base change encoding an Asp27Glu substitution. Immunoblotting with anti-Os(a) and an MAIEA assay with MoAbs to GPA showed that Os(a) is on GPA. Sequencing exon 3 of an Os(a+) person from the only family with Os(a) revealed heterozygosity for a C273T base change encoding a Pro54Ser substitution. A synthetic peptide representing part of GPA with the Os(a) mutation (VRTVYPSEEETGE) completely inhibited anti-Os(a), whereas the control peptide (VRTVYPPEEETGE) did not inhibit anti-Os(a). CONCLUSION: Ny(a) and Os(a) are low-frequency antigens of the MNS blood group system that represent Asp27Glu and Pro54Ser substitutions in GPA, respectively.

Amino Acid Sequence↗

PubMed1999

South-east asian ovalocytic (SAO) erythrocytes have a cold sensitive cation leak: implications for in vitro studies on stored SAO red cells.

South-east Asian ovalocytosis (SAO) results from the heterozygous presence of an abnormal band 3, which causes several alterations in the properties of the erythrocytes. Although earlier studies suggested that SAO erythrocytes are refractory to invasion in vitro by the malarial parasite Plasmodium falciparum, a more recent study showed that fresh SAO cells were invaded by the parasites, but became resistant to invasion on storage because intracellular ATP was depleted more rapidly than normal. Here we show that SAO red cells are much more leaky to sodium and potassium than normal red cells when stored in the cold. This leak was much less marked when the cells were stored at 25 or 37 degreesC. Incubation for 3.5 h at 37 degreesC of cold-stored SAO red cells did not restore sodium and potassium to normal levels, probably because the depleted ATP level in cold-stored SAO red cells is further reduced with incubation at 37 degreesC. The increased leakiness of SAO red cells is non-specific and extends to calcium ions, taurine, mannitol and sucrose. These results suggest that SAO red cells undergo a structural change on cooling. Since many of the reports describing altered properties of SAO red cells have used cells which have been stored in the cold, these results need re-evaluation using never-chilled SAO red cells to assess whether the cells have the same abnormal properties under in vivo conditions.

Adenosine Triphosphate↗

PubMed1999

Stereolithographic biomodelling in cranio-maxillofacial surgery: a prospective trial.

Stereolithographic (SL) biomodelling is a new technology that allows three-dimensional (3-D) computed tomography (CT) data to be used to manufacture solid plastic replicas of anatomical structures (biomodels). A prospective trial with the objective of assessing the utility of biomodelling in complex surgery has been performed. Forty-five patients with craniofacial, maxillofacial, skull base cervical spinal pathology were selected. 3-D CT or MR scanning was performed and the data of interest were edited and converted into a form acceptable to the rapid prototyping technology SL. The data were used to guide a laser to selectively polymerize photosensitive resin to manufacture biomodels. The biomodels were used by surgeons for patient education, diagnosis and operative planning. An assessment protocol was used to test the hypothesis that 'biomodels in addition to standard imaging had greater utility in the surgery performed than the standard imaging alone'. Biomodels significantly improved operative planning (images 44.09%, images with biomodel 82.21%, P < .01) and diagnosis (images 65.63%, images with biomodel 95.23%, P < .01). Biomodels were found to improve measurement accuracy significantly (image measurement error 44.14%, biomodel measurement error 7.91%, P < .05). Surgeons estimated that the use of biomodels reduced operating time by a mean of 17.63% and were cost effective at a mean price of $1031 AUS. Patients found the biomodels to be helpful for informed consent (images 63.53%, biomodels 88.54%, P < .001). Biomodelling is an intuitive, user-friendly technology that facilitated diagnosis and operative planning. Biomodels allowed surgeons to rehearse procedures readily and improved communication between colleagues and patients.

Cephalometry↗

PubMed1999

Glycophorin A mutation Ala65 --> Pro gives rise to a novel pair of MNS alleles ENEP (MNS39) and HAG (MNS41) and altered Wrb expression: direct evidence for GPA/band 3 interaction necessary for normal Wrb expression.

We report here a novel Glycophorin A (GPA) mutation Ala65 --> Pro which gives rise to a low-incidence antigen HAG, lack of a high-incidence antigen ENEP and aberrant expression of the high-incidence Wrb antigen. Anti-ENEP was identified in the serum of a transfused male patient (E.H.) who was homozygous for a GPA Ala65 --> Pro mutation and possessed a novel low-incidence antigen which we have called HAG. An unrelated HAG-positive individual, heterozygous for the Ala65 --> Pro mutation, has also been identified. Anti-HAG was present in several multispecific antisera to low-incidence antigens and in one monospecific serum. Normal expression of the Wrb antigen depends on the presence of amino acid Glu658 of band 3 and on the presence of GPA. However, a specific epitope on GPA has not previously been implicated. DNA sequence analysis of band 3 from patient E.H. was normal in the region of Wra/Wrb polymorphism with homozygous presence of Glu658 and therefore the abnormal Wrb expression results from the Ala65 --> Pro mutation in GPA. The ENEP and HAG antigens have been assigned the MNS blood group system numbers 002.039 and 002.041, respectively, by the ISBT Working Party on Terminology for Red Cell Surface Antigens.

Alanine↗

PubMed1999

Erythroid band 3 variants and disease.

This review describes some of the naturally occurring band 3 (AEI) variants and their association with disease. Southeast Asian Ovalocytic (SAO) band 3, an inactive and misfolded protein, is probably only maintained in certain populations because it provides protection against the cerebral form of malaria. Many mutations that cause instability of band 3, either at the mRNA or protein level, result in hereditary spherocytosis (HS). Some polymorphisms alter amino acid residues in the extracellular loops of band 3 and are associated with blood group antigens. A truncated form of AEI is expressed in kidney cells and certain AEI mutations are associated with distal renal tubular acidosis (dRTA). The molecular basis of these variants and their effect on the structure and function of band 3 are discussed. The association between band 3 and glycophorin A (GPA) and the structure/function changes of band 3 in the absence of GPA are also described.

Acidosis, Renal Tubular↗

PubMed1999

On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed.

The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)6) from an unclarified Escherichia coli homogenate using an expanded bed of Ni2+ loaded STREAMLINE Chelating. The level of GST-(His)6 in the abstracted homogenate samples was measured using Zn2+ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed.

Chromatography, Affinity↗

PubMed1999

The effect of column verticality on separation efficiency in expanded bed adsorption.

The effect of column verticality on liquid dispersion and separation efficiency in expanded bed adsorption columns was investigated using 1 and 5 cm diameter columns. Column misalignment of only 0.15 degree resulted in the reduction of the Bodenstein number from 140 to 50 for the 1 cm dia. column and from 75 to 45 for the 5 cm dia. column. This degree of misalignment was not detectable by visual assessment of adsorbent particle movement within the column. Depending on the relative importance of transport limitations, kinetic limitations and dispersion to any specific separation, this increase in dispersion with column alignment can significantly affect separation efficiency. Pure protein breakthrough profiles resulting from the application of bovine serum albumin onto STREAMLINE Q XL demonstrated that, at 10% breakthrough, 7.8% more protein could be applied to a vertical 1 cm dia. column compared to the same column misaligned by 0.15 degree. When an unclarified yeast homogenate was applied to a 1 cm dia. vertical column packed with STREAMLINE DEAE, 10% breakthrough of glucose-6-phosphate dehydrogenase (G6PDH) corresponded to a load 55% greater compared to the same column aligned 0.185 degree off-vertical. The G6PDH breakthrough curves for vertical and 0.15 degree off-vertical runs performed using a 5 cm column were essentially indistinguishable.

Adsorption↗

PubMed1999

Evaluation of the effect of in-bed sampling on expanded bed adsorption.

An expanded bed adsorption (EBA) column (5 cm diameter) has been modified to allow the abstraction of liquid samples from various positions along the height of an expanded bed. As the adsorbent particles were fluidized, in-bed monitoring of key component concentrations during feedstock application, washing and elution was achieved by the withdrawal of liquid samples from the voids within the expanded bed through ports along the wall of the column. Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line. On-line monitoring can be used to control the duration of the loading stage and as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption. Studies of residence time distributions indicated that the modifications to the column do not significantly affect liquid dispersion. Using the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on Streamline DEAE as a model system, comparison of breakthrough curves for runs when in-bed monitoring was and was not performed also suggested that separation efficiency is not appreciably affected by in-bed sampling.

Adsorption↗

PubMed1998

A novel insertion mutation (1286insC) in exon 9 of the factor XIII-A subunit gene.

Molecular studies have been performed on a Greek family with factor XIII-A subunit deficiency. The 15 exons of the A subunit gene were amplified by polymerase chain reaction and analysed by direct nucleotide sequencing. A homozygous single base insertion (1286insC) in exon 9 of the gene was identified in three affected family members. The insertion results in a frameshift and a premature stop signal a short distance downstream at codon 403. Any A subunit protein expressed is likely to be unstable and lack part of the catalytic core domain together with both beta barrel domains towards the C-terminal of the molecule. This study contributes to our knowledge of the mutational spectrum in patients with factor XIII-A deficiency.

DNA Transposable Elements↗

PubMed1998

The association between familial distal renal tubular acidosis and mutations in the red cell anion exchanger (band 3, AE1) gene.

In distal renal tubular acidosis (dRTA) the tubular secretion of hydrogen ion in the distal nephron is impaired, leading to the development of metabolic acidosis, frequently accompanied by hypokalemia, nephrocalcinosis, and metabolic bone disease. The condition can be familial, when it is usually inherited as an autosomal dominant, though there is a rarer autosomal recessive form associated with nerve deafness. It has been shown that the autosomal dominant form of dRTA is associated with a defect in the anion exchanger (AE1) of the renal collecting duct intercalated cell. This transporter is a product of the same gene (AE1) as the erythrocyte anion exchanger, band 3. In this review we will look at the evidence for this association. Studies of genomic DNA from families with this disorder have shown, both by genetic linkage studies and by DNA sequencing, that affected individuals are heterozygous for mutations in the AE1 gene whilst unaffected family members have a normal band 3 sequence. Mutations have been found in the region of proposed helices 6 and 7 of the membrane domain of band 3 and involve amino acids Arg-589 and Ser-613, and in the COOH-terminal domain of band 3. Studies of red cell band 3 from these families have provided information on the effect these mutations have on the structure and function of erythrocyte band 3. Expression studies of the erythroid and kidney isoforms of the mutant AE1 proteins, in Xenopus laevis oocytes, have shown that they retained chloride transport activity, suggesting that the disease in the dRTA families is not related simply to the anion transport activity of the mutated proteins. A possible explanation for the dominant effect of these mutant AE1 proteins in the kidney cell is that these mutations affect the targeting of AE1 from the basolateral to the apical membrane of the alpha-intercalated cell.

Acidosis, Renal Tubular↗

PubMed1997

Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene.

All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome.

Migration inhibitory factor (MIF) is known to exert significant pro-inflammatory effects and has the potential to override the anti-inflammatory action of glucocorticoids. In this study we have identified significant quantities of MIF in the alveolar airspaces of patients with acute respiratory distress syndrome (ARDS). We show in alveolar cells from patients with ARDS that MIF augments pro-inflammatory cytokine secretion (TNF alpha and IL-8), anti-MIF significantly attenuates TNF alpha and IL-8 secretion and MIF overrides, in a concentration-related fashion, the anti-inflammatory effects of glucocorticoids. These findings suggest that MIF may act as a mediator sustaining the pulmonary inflammatory response in ARDS and that an anti-MIF strategy may represent a novel therapeutic approach in inflammatory diseases such as ARDS.

Cells, Cultured↗

PubMed1996

The low-incidence blood group antigen, Wda, is associated with the substitution Val557-->Met in human erythrocyte band 3 (AE1).

The Waldner blood group antigen (Wda) was first identified in members of a Hutterite kindred. Evidence that the gene governing the Waldner polymorphism is located on chromosome 17, and the observation that the antigen is inactivated by chymotrypsin prompted the investigation of a possible association between Wda and band 3. Single Stranded Conformational Polymorphism (SSCP) analysis and DNA sequence analysis of the AE1 gene, from subjects of known Waldner phenotypes, showed a heterozygous mutation leading to the substitution Val557-->Met in the presumptive Wd(a+) heterozygotes. Therefore the Wda blood group antigen is associated with the presence of Met557 on band 3. the Waldner antigen has been assigned to the Diego blood group system with the International Society of Blood Transfusion number D15.

Blood Group Antigens↗

PubMed1996

Structure-function relationships of band 3 variants.

This review describes many of the naturally occurring band 3 variants that have been reported in the literature to date; from the common band 3 Memphis, to the rare band 3 HT. Both the molecular basis of these variants, and their effect on the structure and/or function of band 3, are described. The blood group antigens that have recently been assigned to band 3, such as Diego, Wright, Waldner, Redelberger and Warrior, are mentioned. Band 3 variants that affect the morphology of the red cell (e.g. acanthocytosis in band 3 HT and stomatocytic ovalocytosis in band 3 SAO) are described, as are many of the band 3 mutations that cause instability, either at the mRNA or protein level, and hence hereditary spherocytosis (HS). Band 3 variants that affect the binding pocket of the anion transport inhibitor, 4,4'-diisothiocyanato-2,2'-dihydrostilbene disulphonic acid (H2DIDS), (e.g. Diego and band 3 HT) and band 3 variants that affect the rate of anion transport (e.g. band 3 HT and band 3 in red cells that lack glycophorin A (GPA)) are reviewed in greater detail. The association between band 3 and GPA is discussed; both with respect to the Wright antigens and with regards the structure/function of band 3 in the absence of GPA.

Acanthocytes↗

PubMed1995

Changes in the blood group Wright antigens are associated with a mutation at amino acid 658 in human erythrocyte band 3: a site of interaction between band 3 and glycophorin A under certain conditions.

The Wright (Wr) blood group antigens, Wra and Wrb, have been suggested to be determined by alleles of the same gene. The Wrb antigen appears to involve both red blood cell (RBC) band 3 and glycophorin A (GPA). We have examined the cDNA sequences of the band 3 and GPA of one of the two known Wr(a+b-) individuals. We show that this individual is homozygous for the mutation Glu658-->Lys in band 3, but has normal GPA. Putative heterozygotes with Wr(a+b+) RBCs have both Glu and Lys at residue 658 of band 3, whereas the common Wr(a-b+) RBC phenotype only have band 3 with Glu658. The Wra and Wrb antigens are determined by the amino acid at residue 658 of band 3 and are antithetical. Examination of the amino acid sequence and Wrb antigen expression of GPA-related hybrid glycophorins suggests that Arg61 of GPA interacts with Glu658 of band 3 to form the Wrb antigen. We suggest that the interaction is stabilized by the presence of anti-Wrb antibodies and that this site of association between GPA and band 3 may be responsible for the previously reported ability of anti-GPA antibodies to decrease the deformability of RBCs.

Alleles↗

PubMed1994

Altered band 3 structure and function in glycophorin A- and B-deficient (MkMk) red blood cells.

The anion transport activity of the human erythrocyte anion transporter (band 3; AE1) has been examined in both normal and glycophorin A (GPA)-deficient (MkMk) human red blood cells (RBCs). The sulfate transport activity of MkMk cells (from two ethnically diverse sources) was approximately 60% that of normal erythrocytes under the transport assay conditions used. However, MkMk and normal RBCs contained similar amounts of band 3. The reduction in sulfate transport activity was shown to be caused by an increase in the apparent Km for sulfate in MkMk RBCs, suggesting the band 3 in the MkMk RBCs has a lowered binding affinity for sulfate anions. The size of the N-glycan chain on band 3 of the MkMk cells was larger than that on band 3 from normal RBCs. In contrast, the size of the N-glycan chain on the glucose transporter (GLUT1) from MkMk cells was smaller than that on GLUT1 from normal cells. The possible role of GPA in the biosynthesis and anion transport activity of band 3 in normal RBCs is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Band 3 Memphis variant II. Altered stilbene disulfonate binding and the Diego (Dia) blood group antigen are associated with the human erythrocyte band 3 mutation Pro854-->Leu.

Band 3 Memphis is a commonly occurring polymorphic form of the human red cell anion transporter (band 3, AE1). Band 3 Memphis migrates more slowly on an SDS-polyacrylamide gel than normal band 3 and results from a point mutation Lys56-->Glu. Two types of band 3 Memphis, variants I and II, can be distinguished by their susceptibility to covalent labeling with H2DIDS (4,4'-diisothiocyanato-2,2'-dihydrostilbene disulfonate). Memphis variant II is more readily labeled than Memphis variant I or normal band 3. The Memphis variant II is also associated with the presence of the Diego (Dia) blood group antigen on the red cells. We have shown that Memphis variant II carries the polymorphism Pro854-->Leu, as well as Lys56-->Glu. The blood group antigen (Dia) present at the surface of Memphis variant II type red cells suggests the mutation Pro854-->Leu causes a change in the structure of an extracellular loop of Memphis variant II band 3. We discuss possible ways in which the mutation Pro854-->Leu affects the reactivity of Lys539 to covalent reaction with H2DIDS.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗