Biomedical subjects

C J Chapman

Publications and source records attributed to C J Chapman.

At least 19 recordsLinked to original sources

PubMed2005

Malignancy-induced autoimmunity to MUC1: initial antibody characterization.

Numerous reports document the existence of autoantibodies to MUC1 in the circulation of individuals with breast and other solid malignancies, with the majority of researchers utilizing MUC1 peptides in their detection. This report documents the purification, using peptide and whole molecule, and characterization of such autoantibodies from an individual with an unusual, highly MUC1-positive, myosarcoma. Purification of autoantibodies from serum was performed using affinity chromatography against either MUC1 peptide or whole molecule MUC1 [derived both from the patient (Pt-MUC1) and from a pool of sera from patients with advanced breast cancer (ABC-MUC1)]. Enzyme-linked immunosorbent assays (ELISAs) were used to compare specificity of purified autoantibodies. Peptide epitopes were determined by Ptifcan system against 7-mer peptides covering the 20 amino acid repeat of the MUC1 extracellular domain. Substantially higher amounts of autoantibodies were isolated when purifying against Pt-MUC1 rather than either ABC-MUC1 or peptide. Whole molecule purified autoantibodies demonstrated an increased specificity for tumour-derived MUC1. Pt-MUC1 autoantibodies were of both the immunoglobulin (Ig)G and IgM class, whilst autoantibodies purified against ABC-MUC1 and MUC1 peptide were IgG only. A greater range of peptide epitopes was defined by those autoantibodies purified against whole molecule. This report presents data indicating the presence of autoantibodies to MUC1 in an individual diagnosed with a MUC1 over-expressing myosarcoma. Confirmation of these autoantibodies as being specific for tumour-associated MUC1 is given. Further, it suggests that, although autoantibodies are present that recognize core protein determinants, the initial, and dominant, immunizing epitope is not purely pretentious in nature.

Antibody Specificity↗

PubMed2001

Transport properties of the calcium ionophore ETH-129.

The transport mechanism and specificities of ionophore ETH-29 have been investigated in a highly defined phospholipid vesicle system, with the goal of facilitating the application of this compound to biological problems. ETH-129 transports Ca(2+) via an electrogenic mechanism, in contrast to A23187 and ionomycin, which function in a charge neutral manner. The rate of transport is a function of membrane potential, increasing by 3.9-fold per 59 mV over a broad range of that parameter. Rate is independent of the transmembrane pH gradient and strongly stimulated by the uncoupler carbonyl cyanide m-chlorophenylhydrazone when no external potential has been applied. The effect of uncoupler reflects the collapse of an opposing potential arising during Ca(2+) transport, but also reflects the formation of a mixed complex between the uncoupler, ETH-129, and Ca(2+) that readily permeates the vesicle membrane. Oleate does not substitute for the uncoupler in either regard. ETH-129 transports polyvalent cations according to the selectivity sequence La(3+) > Ca(2+) > Zn(2+) approximately equal to Sr(2+) > Co(2+) approximately equal to Ni(2+) approximately equal to Mn(2+), with the magnitude of the selectivity coefficients reflecting the cation concentration range considered. There is little or no activity for the transport of Na(+), K(+), and Mg(2+). These properties suggest that ETH-129 will be useful for investigating the consequences of a mitochondrial Ca(2+) overload in mammalian cells, which is difficult to pursue through the application of electroneutral ionophores.

Biological Transport↗

PubMed2000

Autosomal recessive cerebellar ataxia with oculomotor apraxia (ataxia-telangiectasia-like syndrome) is linked to chromosome 9q34.

Ataxia with oculomotor apraxia (ataxia-telangiectasia-like syndrome [AOA]; MIM 208920) is an autosomal recessive disorder characterized by ataxia, oculomotor apraxia, and choreoathetosis. These neurological features resemble those of ataxia-telangiectasia (AT), but in AOA there are none of the extraneurological features of AT, such as immunodeficiency, neoplasia, chromosomal instability, or sensitivity to ionizing radiation. It is unclear whether these patients have a true disorder of chromosomal instability or a primary neurodegenerative syndrome, and it has not been possible to identify the defective gene in AOA, since the families have been too small for linkage analysis. We have identified a new family with AOA, and we show that the patients have no evidence of chromosomal instability or sensitivity to ionizing radiation, suggesting that AOA in this family is a true primary cerebellar ataxia. We have localized the disease gene, by linkage analysis and homozygosity mapping, to a 15.9-cM interval on chromosome 9q34. This work will ultimately allow the disease gene to be identified and its relevance to other types of autosomal recessive cerebellar ataxias to be determined.

Acid Anhydride Hydrolases↗

PubMed2000

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity.

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.

Aminoquinolines↗

PubMed1999

A human monoclonal antibody encoded by the V4-34 gene segment recognises melanoma-associated ganglioside via CDR3 and FWR1.

A heterohybridoma cell line producing the human monoclonal antibody (MoAb) MDT.1 has been established. The heavy chain of MoAb MDT.1 is encoded by the VH gene segment V4-34 (previously designated VH4-21), and the light chain is encoded by the V kappa 1-L12a gene segment, both in germline configuration. MDT.1 has reactivity against lipid A, double- and single-stranded DNA, red blood cell associated i antigen, and ganglioside antigens. In a panel of tumour cell lines, MDT.1 reacted specifically with melanoma cells and other tumour cells of neuroectodermal origin. Cellular recognition appears to be via tumour-associated ganglioside antigens, and may involve the minimal essential epitope NeuNac alpha 2-->3Gal beta 1-->-4Glc-. Binding to ganglioside antigen is inhibited by the monoclonal anti-idiotypic antibody 9G4. Since the 9G4 idiotope is located in framework region 1 (FWR1) of V4-34-encoded antibodies, this region is likely to be involved, either directly or indirectly, in ganglioside binding. The complementarity-determining region 3 (CDR3) of MDT.1 is arginine rich, with five out of 12 residues being arginine and these residues are candidates for interaction with the negatively charged ganglioside. The ability of MoAb MDT.1 to recognise ganglioside antigens is associated with potentially useful anti-tumour activity.

Amino Acid Sequence↗

PubMed1998

Clonally related IgE and IgG4 transcripts in blood lymphocytes of patients with asthma reveal differing patterns of somatic mutation.

Isotype switching to IgE contributes to atopic asthma, therefore strategies to divert this process to alternative isotypes could have therapeutic relevance. It is known that patients with allergic disease have serum IgE and IgG4 antibodies with similar specificities, and that cytokines such as IL-4 mediate switching to both of these isotypes. Availability of variable region gene analysis has allowed us to probe isotype variants at the single-cell level. An earlier report described identification in a single atopic patient of short transcripts with a common complementarity-determining region "clonal signature" in combination with C mu, C gamma4 and C epsilon. We have extended this analysis, and have identified V(H)-Cgamma4 transcripts with clear clonal relationship to IgE-derived sequences in blood lymphocytes from three of four patients with atopic asthma. No other IgG subclasses were detected, confirming the link between IgE and IgG4. Full sequences were obtained from each clonally related isotype in all patients, and showed extensive somatic mutation. As previously found for IgE, the IgG4 isotypes had evident intraclonal variation. There were shared mutations between isotypes, but also many differences, indicative of separate cell populations with divergent mutational histories. These findings indicate that, in atopic patients, an individual B cell commonly switches to either IgE or IgG4. Cells producing each isotype then co-exist in the recirculating pool, and the balance between them may influence the disease process.

Amino Acid Sequence↗

PubMed1998

Immunogenetic analysis of the heavy chain variable regions of IgE from patients allergic to peanuts.

Peanuts are one of the most allergenic of the foods, and hypersensitivity responses to peanut allergens can be fatal. Although the nature of the antigenic components of peanuts is being defined at the molecular level, there is little information on the induced IgE antibodies, which are central to the allergic reaction. Recognition sites of IgE antibody molecules arise from the variable regions of heavy and light chains (VH and VL). By using nested polymerase chain reactions with specific primers for the available repertoire of VH genes, together with primers in the constant epsilon region, we have amplified VH sequences of IgE from blood lymphocytes of two patients with peanut allergy. After cloning and sequencing the products, we found a predominance of VH1 family use in both patients, which was not found in control IgM-specific primers. The IgE VH sequences were highly somatically mutated, but in only six of 17 cases was there clear evidence for clustering of amino acids indicative of antigen selection. Previous results from patients with allergy to house dust mites have indicated predominance of VH5 use and little evidence for antigen selection. Although results from two patients allergic to peanuts must be regarded as preliminary, they do suggest that the IgE response to peanuts may have a different VH bias, with a similar mutational pattern.

Adolescent↗

PubMed1998

Immunogenetic analysis of a panel of monoclonal IgG and IgM anti-PDC-E2/X antibodies derived from patients with primary biliary cirrhosis.

BACKGROUND/AIMS: Autoantibodies with specificity for the E2 component of the pyruvate dehydrogenase complex (PDC-E2) are commonly present in primary biliary cirrhosis. The aim of this study was to generate and characterise human anti-PDC-E2 monoclonal antibodies and analyse immunoglobulin gene usage and mutation for clues to pathogenesis. METHODS: Peripheral B-lymphocytes from two patients with primary biliary cirrhosis were used to generate heterohybridomas secreting PDC-E2 specific monoclonal antibodies. The antibodies were characterised by ELISA, immunoblotting, indirect immunofluorescence and enzyme inhibition techniques, and their encoding immunoglobulin genes were amplified, cloned and sequenced. RESULTS: Four IgGlambda and one IgMlambda monoclonal antibodies specific for PDC-E2 were generated: all gave bands at 74 kD and 52 kD on PDC immunoblots, two clones were specific for the lipoylated inner lipoyl domain, and all inhibited target enzyme function. Sequence analysis suggested unrestricted VH gene usage, but a strong preference for lambda light chains. The extent of somatic mutation was high (3-20%), with evidence for antigen selection in 3/5 VH sequences. CONCLUSIONS: These monoclonal antibodies closely resemble the hallmark autoantibodies of primary biliary cirrhosis. Their specificities demonstrate true cross reactivity between an epitope on PDC-E2 and Protein X, and the existence of a subset of B cells that recognise only the lipoylated form of the antigen. The pattern of immunoglobulin gene mutations suggests an antigen-driven selection of high affinity IgG autoantibodies, supporting a possible role for exogenous antigen in the pathogenesis of primary biliary cirrhosis.

Amino Acid Sequence↗

PubMed1998

V(H) gene analysis of Burkitt's lymphoma in children from north-western Iran.

Cases of Burkitt's lymphoma (BL) from north-western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional V(H) genes were amplified from 6/12 of the tumour masses and all of these were derived from the V(H)3 family, with 4/6 being derived from the most commonly used V(H)3 family member, V3-23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated V(H) sequences showed a significant clustering of replacement mutations in the complementarity-determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in V(H) sequences of EBV+ve endemic BL (eBL) and EBV-ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, VH gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL.

Amino Acid Sequence↗

PubMed1998

Sequence analysis of V(4-34)-encoded antibodies from single B cells of two patients with systemic lupus erythematosus (SLE).

SLE is an autoimmune disease characterized by the presence of autoantibodies against double-stranded (ds)DNA. A large proportion (approx. 40%) of patients with lupus also have increased levels of serum immunoglobulin encoded by the V(4-34) heavy chain gene, which often fluctuate with disease activity, and this gene is utilized by a subset of anti-dsDNA antibodies. In order to probe the nature of the V(4-34)-encoded immunoglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gene product. Following cell picking, single-cell polymerase chain reaction (PCR) amplification of cDNA was used to investigate both V(H) and V(L) genes. Sequences were obtained from B cells synthesizing IgM (n = 10), IgG (n = 4) and IgA (n = 1). For V(H), all were derived from V(4-34) as expected, and the isotype-switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, V(L) (12 kappa and 3 lambda) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated V(H) sequences were associated with unmutated V(L) sequences. Analysis of the distribution of mutations revealed only minor clustering in complementarity-determining regions (CDRs) characteristic of antigen selection. The CDR3 lengths of V(H) ranged from five to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed repertoire. Basic amino acids were also found at the V(L)-J(L) junctions in 4/15. These findings provide insight into the V(4-34)-V(L) gene combinations used by B cells in patients with SLE which might have clinical relevance.

Adolescent↗

PubMed1998

Insight into Burkitt's lymphoma from immunoglobulin variable region gene analysis.

Analysis of usage of V(H) and V(L) genes, and the degree and pattern of somatic mutation, has been used to investigate the cell of origin and clonal history in cases of Burkitt's lymphoma (BL). Tumor cell lines and biopsy material from patients with endemic, sporadic and AIDS-associated BL have been compared. V(H) genes were most commonly derived from the V(H)3 (52%) and V(H)4 (39%) families. This shows a similar gene usage of the V(H)3 family to that seen in the normal peripheral blood repertoire (55%), but a biased usage of the V(H)4 family (22% in normal). There was no restriction in V(L) gene usage. This overall distribution was similar in all subsets of BL. In all categories, there was significant somatic mutation in both V(H) and V(L) sequences. There was no evidence for accumulation of mutations in cell lines cultured in vitro indicating that all mutations in BL-derived cell lines have accumulated in vivo. The mean percentage level of mutation +/- standard deviation was greater in endemic BL (V(H) = 7.7 +/- 4.0, V(L) = 5.3 +/- 2.2) and AIDS-associated BL (V(H) = 7.5 +/- 3.6, V(L) = 3.9 +/- 1.9) than in sporadic BL (V(H) = 4.0 +/- 2.5, V(L) = 2.2 +/- 1.2). The pattern of somatic hypermutation was similar in V(H) and V(L) sequences of the different types of BL although the light chain genes were less mutated. Mutational patterns in the V(H) genes did not reveal a conventional role for antigen in selection of tumor cell sequences in 23/25 V(H) genes analysed. In contrast, patterns in V(L) sequences were consistent with a role for antigen in 8/13 sBL +/- eBL cases and 8/17 cases overall. The presence of EBV did not seem to influence the quantity or pattern of somatic mutations. Evidence for intraclonal variation was seen in uncloned cell lines from cases of eBL and AIDS-associated BL and confirmed in biopsy material in some, but not all cases of eBL, sBL and AIDS-associated BL examined. These common features indicate that the B-cells involved in all types of BL are derived from cells that have traversed the germinal centre, and that the somatic mutation mechanism may still be operative following neoplastic transformation. Overall, in 10/30 cases, there was evidence of significant clustering of replacement amino acids, in CDRs, particularly in V(L), indicating that the B-cell of origin is likely to have been selected by antigen.

Burkitt Lymphoma↗

PubMed1997

Pattern of usage and somatic hypermutation in the V(H)5 gene segments of a patient with asthma: implications for IgE.

The V(H)5 family contains two functional genes, V5-51 and V(H)32, and appears to be over-represented in IgE antibodies from patients with allergic disease. Previous sequence analysis of V(H)5 gene segments in IgE has revealed a substantial level of somatic hypermutation, with evidence for hotspots. To assess characteristics of V(H)5 gene behavior, V(H)5 gene segments in combination with C mu, C gamma, C alpha, and C epsilon have been amplified from blood B lymphocytes of a patient with atopic asthma. Sequence analysis revealed strong preferential usage of one of the two V(H)5 gene segments (V5-51) by IgM, IgG, and IgA. In contrast, IgE used both genes equally. Levels of somatic mutation were higher following all isotype switches, particularly to IgA. Mutational hotspots were identifiable in all isotypes, leading to several common replacement amino acids. The dominant mutational site in IgM was a common hotspot at Ser31. IgG, IgA, and IgE-derived sequences had mainly common hotspots, with few distinct sites. The results indicate that mutational hotspots are a feature of the V(H)5 gene, are identifiable at an early stage of somatic hypermutation, and are not a unique feature of IgE. Generation of IgE antibodies appears to involve three processes: the preferential use of V(H)5 genes, consistent with superantigen stimulation; the accumulation of somatic mutations in common hotspots, some of which are in complementarity-determining regions (CDR); and the acquisition of non-hotspot mutations in CDR, accounting for approximately 50% of replacement amino acids in these sites, and presumably contributing to affinity maturation.

Adult↗

PubMed1997

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies, II.

We have previously described complement-independent killing of human B lymphocytes by two IgM MoAbs derived from the VH4-34 (VH4.21) gene. Analysis of 17 independently derived VH4-34-encoded MoAbs shows that B cell toxicity is not limited to the two described MoAbs, but is a general property shared by a subset of MoAbs derived from the VH4-34 gene. As observed by two independent microscopy techniques, giant membrane pores were formed on target B cells within 10-15 min of exposure to cytotoxic VH4-34-derived MoAbs. Toxicity by individual MoAb correlated directly to its B cell binding intensity measured by FACS, i.e. stronger the binding greater the killing. Sequence analysis showed that V(H) region in germ-line or in near germ-line configuration was necessary but not sufficient for B cell binding. In addition, a particular sequence motif enriched in basic amino acids in the CDR3 may be required to supplement the reactivity mediated by the V(H) region of the MoAb molecule. VH4-34-encoded antibodies that fulfil the above sequence requirements have cold agglutinin activity towards the i antigen of cord erythrocytes. In vivo, such anti-i/anti-B cell antibodies are rarely detected in healthy adults, but serum levels are dramatically elevated in selective pathological conditions, such as systemic lupus erythematosus and infectious mononucleosis. This strict regulation may be related to the novel and rapid mechanism of human B cell toxicity demonstrated by antibodies encoded by a single human V(H) gene.

Adult↗

PubMed1997

Phage surface expression for analysis of recognition sites of human autoantibodies: comparison of single chain Fv and Fab.

Human autoantibodies can mediate the pathology of autoimmune disease. To plan therapeutic intervention based on inhibition of binding to autoantigen, it is desirable to map the amino acid residues involved in recognition. Bacterial expression provides a simple and rapid system for production and mutagenesis of recombinant antibody fragments, but there are problems with reproducible folding and dispersity of soluble products. Expression at the surface of phage particles avoids both these problems, and allows variable region gene sequences to be expressed as functional monomers. To apply this technology, we have expressed a human anti-DNA monoclonal antibody, derived from a hybridoma, at the surface of the phage. The presence of the phage particle allowed highly sensitive detection of antibody activity by ELISA, so that minimal levels of expression were required. By measuring the amount of expressed antibody protein, it was possible to analyze the antibody activity/microgram. We expressed the antibody as FAB or single chain (sc) Fv, and found binding activities to be closely similar. The parental anti-DNA antibody utilizes the human V4-34 gene and displays the associated conformation-dependent 9G4 idiotope. Both the Fab and scFv expressed the idiotope, confirming similar molecular folding. This technology can be applied rapidly and efficiently to investigations of human autoantibody structure, with the option for expression as either Fab or scFv, and the only requirement being knowledge of VH and VL sequences.

Antibodies, Anti-Idiotypic↗

PubMed1996

VH and VL gene analysis in sporadic Burkitt's lymphoma shows somatic hypermutation, intraclonal heterogeneity, and a role for antigen selection.

Tumor cell lines and one tumor biopsy from seven cases of Epstein-Barr virus (EBV) genome-negative sporadic Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig variable region genes. The VH genes were derived from the VH 3 (one) and VH4 (six) families and both the IgM-positive (six) and the IgA-positive (one) were all mutated from their germline counterparts. The VL genes were derived from V kappa 1 (one), V kappa 3 (one), V lambda 1 (four), and V lambda 2 (one) families and were also somatically hypermutated. Biopsy material from one of the IgM-positive cases showed VH and VL sequences that matched the derived cell line, with additional intraclonal sequence heterogeneity, indicating that the tumor cells had undergone posttranformation somatic mutation. Mutational patterns in V(H) genes did not show a conventional role for antigen in selecting tumor cell sequences. In contrast, patterns in VL sequences were consistent with a role for antigen in five of seven cases. The pattern of extensive scattered somatic hypermutation and intraclonal variation is similar to that in VH sequences of EBV genome-positive endemic BL, although the degree of mutational activity is less. These common features indicate that B cells involved in the two variants of BL may share a common clonal history.

Adolescent↗

PubMed1996

Ionophore 4-BrA23187 transports Zn2+ and Mn2+ with high selectivity over Ca2+.

The cation transport selectivities of the Ca2+ ionophores A23187, Ionomycin, and 4-BrA23187 have been determined using a model system comprised of phospholipid vesicles loaded with the chelator/indicator Quin-2. At pH 7.00 and a 100 microM concentration of the cations, A23187 displays the transport selectivity sequence Zn2+ > Mn2+ > Ca2+ > Co2+ > Ni2+ > Sr2+, with the absolute rates of transport spanning approximately 3 orders of magnitude. Similar data are obtained with Ionomycin, although the relative transport rates of Zn2+ and Mn2+ are equivalent, and the range of absolute rates is decreased by a factor of approximately 3. When values are normalized to those of Ca2+, transport selectivity is seen to be only weakly related to complexation or extraction selectivity. It is also seen that, when used to manipulate Ca2+ (or Mg2+), both ionophores can be expected to alter the distribution of additional divalent cations which have known biological activities. 4-BrA23187 is a low-activity ionophore for Ca2+, compared to A23187 and Ionomycin, while retaining comparable activities as an ionophore for the other cations. As a consequence, 4-BrA23187 is highly selective for the transport of Zn2+ and Mn2+, compared to Ca2+, with selectivity ratios approaching that of valinomycin for K+ over Na+ when conditions are optimal. Plots of the log of the rate of cation transport vs the log of the ionophore concentration indicate that Ca2+ is transported primarily as a 2:1 complex by A23187 and 4-BrA23187, but Zn2+ and Mn2+ are transported, in part, as 1:1 complexes. These findings, together with a postulated low stability of 2:1, compared to 1:1 complexes between 4-BrA23187 and divalent cations, partially explain the novel transport selectivity of this compound. Unlike A23187 or Ionomycin, 4-BrA23187 may be useful for investigating cell regulation by Zn2+ and Mn2+, without interference by regulatory mechanisms which respond to Ca2+.

Aminoquinolines↗

PubMed1996

Use of phage surface expression to analyze regions of human V4-34(VH4-21)-encoded IgG autoantibody required for recognition of DNA: no involvement of the 9G4 idiotope.

The V4-34 gene encodes the majority of autoanti-red cell Abs of I/i specificity. It also encodes a proportion of autoanti-DNA Abs found in patients with systemic lupus erythematosus. Nucleotide sequence analysis of mAbs that use this gene has indicated a role for CDR3 in discrimination between these autoantigens. Specifically, anti-DNA activity may require basic amino acids, such as arginine, found in this region. To investigate this requirement, we have expressed VH and VL sequences from a patient's IgG anti-DNA mAb, as Fab molecules at the surface of phage. Expressed Fab bound strongly to DNA, whereas control VH and VL pairs from an anti-red cell mAb did not. Replacement of the homologous mutated V4-34 sequence by germ-line sequence did not affect binding, indicating that somatic mutations in VH did not contribute significantly. In contrast, replacement of the basic CDR3 by an anti-red cell CDR3 abrogated anti-DNA activity, confirming its major role. However, an influence of VL was revealed by replacing homologous mutated V kappa IIIb by an unmutated V kappa IIIb sequence, reducing binding by approximately 50%. This influence was apparent only with homologous VH since the mutated V kappa was unable to generate anti-DNA activity when combined with anti-red cell VH. The 9G4 idiotope, which arises from FWR1, was expressed by all constructs. Substitution of Trp by Ser at position 7 in FWR11 caused complete loss of idiotope expression, with no effect on recognition of DNA, indicating no influence of idiotope expression on anti-DNA activity. Phage surface expression provides a powerful and rapid technique for assessing sequences relevant for Ab specificity or idiotope expression.

Amino Acid Sequence↗

PubMed1996

On the use of fluorescent probes to estimate free Mg2+ in the matrix of heart mitochondria.

The fluorescent probe furaptra shows increases and decreases in the concentration of free magnesium ion, [Mg2+], in the mitochondrial matrix with changes in total Mg2+ and ligand availability. The factors involved in the calibration of these fluorescence changes in terms of absolute [Mg2+] have been investigated. The affinity of furaptra for Mg2+ is highly dependent on both temperature and ionic strength. The Kd for Mg-furaptra in solution in 100 mM KCl was found to be 2.1 +/- 0.1 mM at 25 degrees C. The use of this Kd to calculate matrix [Mg2+] is more reliable than in situ Kd measurements because ionophores, such as BrA23187 and ionomycin, do not equilibrate external Mg2+ with the matrix in an acceptable way. Furaptra is present at high concentrations (up to 500 microM) in the matrix when introduced by hydrolysis of the acetoxymethyl ester. However, absorbance spectra of aqueous solutions show no evidence of dimerization of the probe or other changes in properties at these concentrations. Fluorescence intensity at 340 nmex is strongly attenuated for matrix-sequestered furaptra, mag-fura-5, and mag-indo-1. This appears to result in part from preferential binding of the Mg-probe to mitochondrial proteins. The fluorescence of uncomplexed furaptra at 375-380 nmex seems unaffected by protein binding, however, and changes in intensity in this region of the spectrum can be used in conjunction with the Kd found in aqueous solution to estimate matrix [Mg2+]. The presence of secondary equilibria, such as protein binding, and possible changes in ionic strength may undermine exact quantitation by this method. However, values for matrix [Mg2+] obtained in this way (0.5 to 0.7 mM) correspond well to estimates by other available methods and each of these methods suffers from comparable uncertainties.

Animals↗