Search PubMed⌕ Search

Biomedical subjects

J D Capra

Publications and source records attributed to J D Capra.

At least 55 records · Page 3Linked to original sources
PubMed1995

Directional cloning of an oligonucleotide fragment into a single restriction site.

Oligonucleotide fragments can be directionally subcloned into vectors at a single restriction site. By using T4 DNA polymerase exonuclease activity to treat vector DNA, single-stranded ends can be generated. The oligonucleotide sequences are designed to have sequence complementary to these single-stranded ends. Through the homologous annealing of oligonucleotides to the treated vector ends, the successfully subcloned molecules forms a circular recombinant DNA that is ready for transformation. There is no sequence restriction at the ends of the DNA fragment. All restriction site ends are accessible to this method. This approach for oligonucleotide fragment insertion and together with our previously described general method of exonuclease induced DNA subcloning provide convenient methods for the construction of recombinant DNA.

Amino Acid Sequence↗
PubMed1995

The molecular structure of human antibodies specific for the human immunodeficiency virus.

The molecular structure of human antibodies that are specific for human immunodeficiency virus-1 (HIV-1) are of increasing interest as AIDS research progresses toward passive immunotherapeutics in the maintenance and prevention of infection. In recent years a number of human, HIV-specific hybridomas and EBV-transformed B cell lines, as well as a combinatorial library, have been developed and characterized at the molecular level. These sources have provided valuable information on the immunoglobulin heavy- and light-chain variable-region gene usage and the extent and appearance of somatic mutation in a disease where the immune system is under constant stimulation over a long period of time. In this article we review the current data available on the molecular structure of these antibodies.

Amino Acid Sequence↗
PubMed1995

The human antibody repertoire: heavy and light chain variable region gene usage in six alloantibodies specific for human HLA class I and class II alloantigens.

Peripheral blood B lymphocytes have been isolated from healthy individuals who were immunized with lymphocytes from HLA-incompatible donors and transformed with Epstein-Barr virus to produce human monoclonal cell lines specific for human HLA molecules. The cell lines have been previously characterized and are known to bind to various class I and class II alloantigens. In this report we describe the molecular characterization of the heavy and light chain variable region gene segments that are utilized by these monoclonal antibodies. Using the polymerase chain reaction and primer pairs specific for the respective constant region and VH or VL family, rearranged variable region gene segments were amplified from cDNA from individual cell lines. Products were then subcloned, sequenced and analysed for gene usage and apparent somatic mutation. The results show that the VH3 gene family predominates in a group of six heavy chains (four out of six) with one VH1 and one VH4 gene segment. The light chain variable region gene family usage is more diverse with 2 V kappa 3, 1 V kappa 1, 2 V lambda 2 and 1 V lambda 3. The extent of apparent somatic mutation is minimal, relative to our previous observations in a group of high affinity human monoclonal antibodies specific for pathogenic organisms.

Amino Acid Sequence↗
PubMed1995

TAP2 association with insulin-dependent diabetes mellitus is secondary to HLA-DQB1.

IDDM is known to be associated with genes of HLA complex, particularly alleles of HLA-DQ. The 40-kb TAP gene complex is located approximately 150 kb centrometric to the DQB1 locus. The TAP1-TAP2 protein heterodimer is required for normal expression levels of class I, molecules on the surface of cells. While present evidence implicates HLA-DQ as the major susceptibility locus in IDDM, as class I expression apparently plays a role in the progression of disease, the possibility exists that the association attributed to HLA-DQ is in fact due to an association with the TAP genes. Several studies have concluded that the alleles of TAP1 are not significantly associated with IDDM; this report concentrates on the more telomeric TAP2 locus. During this investigation, six previously described TAP2 alleles were identified in 208 normal Caucasians and 241 Caucasian diabetics. Sequence analysis of cDNA clones identified a seventh allele of TAP2, TAP2*F, which contains an arginine-to-cystine interchange at amino acid position 651. Overall, our results indicate only a modest association of IDDM with TAP2; however, the newly described TAP2*F allele was found to be significantly increased in a modest subset of our large diabetic population. These data, generated from the same population of controls and diabetics we previously studied at all other relevant MHC loci, provide additional evidence that the HLA susceptibility to IDDM maps to HLA-DQ.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Production and characterization of recombinant human Ku antigen.

Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo. It was originally described as an autoantigen in patients with certain autoimmune diseases. The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful. Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer. In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA. Neither subunit binds DNA by itself indicating that heterodimerization is essential for function. We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag. The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.

Animals↗
PubMed1994

Recombinant human IgA expressed in insect cells.

IgA serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities in serum. To map the sites of interaction of immune effector molecules with the IgA constant region (C alpha), we have expressed soluble, chimeric human IgA in insect cells using recombinant baculoviruses. This antibody is correctly assembled into heavy chain/light chain heterodimers, N-glycosylated, and secreted by the insect cells; further, when coexpressed with a human J chain, the antibodies can assemble into dimers. The recombinant protein is authentic by a number of criteria, including antigen-binding, recognition by monoclonal antibodies, complement fixation via the alternative pathway, and specific binding to the monocyte IgA Fc receptor. We have also constructed viruses which encode structurally altered IgA heavy chains. Using one of these variant viruses, we have shown that glycosylation of the second domain of C alpha is required for interaction with the monocyte IgA Fc receptor. This system should prove useful in further characterization of the structure-function relationships in human C alpha.

Animals↗
PubMed1994

Analysis of somatic mutation in five B cell subsets of human tonsil.

Using a series of phenotypic markers that include immunoglobulin (Ig)D, IgM, IgG, CD23, CD44, Bcl-2, CD38, CD10, CD77, and Ki67, human tonsillar B cells were separated into five fractions representing different stages of B cell differentiation that included sIgD+ (Bm1 and Bm2), germinal center (Bm3 and Bm4), and memory (Bm5) B cells. To establish whether the initiation of somatic mutation correlated with this phenotypic characterization, we performed polymerase chain reaction and subsequent sequence analysis of the Ig heavy chain variable region genes from each of the B cell subsets. We studied the genes from the smallest VH families (VH4, VH5, and VH6) in order to facilitate the mutational analysis. In agreement with previous reports, we found that the somatic mutation machinery is activated only after B cells reach the germinal center and become centroblasts (Bm3). Whereas 47 independently rearranged IgM transcripts from the Bm1 and Bm2 subsets were nearly germline encoded, 57 Bm3-, and Bm4-, and Bm5-derived IgM transcripts had accumulated an average of 5.7 point mutations within the VH gene segment. gamma transcripts corresponding to the same VH gene families were isolated from subsets Bm3, Bm4, and Bm5, and had accumulated an average of 9.5 somatic mutations. We conclude that the molecular events underlying the process of somatic mutation takes place during the transition from IgD+, CD23+ B cells (Bm2) to the IgD-, CD23-, germinal center centroblast (Bm3). Furthermore, the analysis of Ig variable region transcripts from the different subpopulations confirms that the pathway of B cell differentiation from virgin B cell throughout the germinal center up to the memory compartment can be traced with phenotypic markers. The availability of these subpopulations should permit the identification of the functional molecules relevant to each stage of B cell differentiation.

Antigens, CD↗
PubMed1994

Biased utilization of DHQ52 and JH4 gene segments in a human Ig transgenic minilocus is independent of antigenic selection.

We have assessed the effect of antigenic selection on human Ig heavy chain D and JH gene segment utilization in mice that contain transgenes composed of 2 VH (psi VH3-105 and VH5-251), 10 D, 6 JH, C mu, and C gamma 1 human gene segments. Human heavy chains using the functional VH5-251 gene segment are expressed in the serum and on the surface of murine B cells. The second VH gene segment (psi VH3-105) is not expressed as a protein but is rearranged and transcribed into mRNA. We previously reported that the functional VH5-251 mu transcripts preferentially used the DHQ52 and JH4 gene segments similar to their use in the human repertoire. Here, we demonstrate that the nonfunctional (psi VH3-105) gene segment shows the same bias in D and JH gene segment utilization. Because transcripts using the pseudo-VH gene segment cannot be subjected to antigenic selection, we conclude that the restricted repertoire observed is Ag independent. Analysis of pseudo VH gene segment recombination products reveals no bias in D gene segment reading frame utilization. Finally, we demonstrate that in the human transgene coding sequence complementarities can affect the recombination site and that D inversion is common.

Animals↗
PubMed1994

Nucleotide sequence analysis of natural and combinatorial anti-PDC-E2 antibodies in patients with primary biliary cirrhosis. Recapitulating immune selection with molecular biology.

We have analyzed at the nucleotide level the variable region gene sequences of five human mAbs and five recombinant Fab fragments derived from the mesenteric lymph nodes of patients with primary biliary cirrhosis. Both mAbs and Fabs were monospecific for dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex, which has been shown to be the major autoantigen of primary biliary cirrhosis. We found that although the mAbs, mainly of the IgM isotype, were encoded by a diverse array of VH and VL gene segments either as direct copies of germline genes or somatically mutated, the recombinant IgG Fabs expressed clonally related heavy chains displaying a high number of somatic mutations that very likely occurred in the context of Ag selection. Combinatorial pairing of clonally related heavy chains with highly homologous light chains suggests that the IgG anti-pyruvate dehydrogenase complex repertoire of primary biliary cirrhosis patients is the result of the clonal expansion of a restricted set of B cells.

Amino Acid Sequence↗
PubMed1994

Calreticulin is released from activated neutrophils and binds to C1q and mannan-binding protein.

The Ca2+ storage protein calreticulin is associated with the endoplasmic reticulum and shares a high degree of amino acid homology with the surface receptor C1q-R. In this study, flow cytometric analysis detected calreticulin on the neutrophil surface, which decreased during stimulation probably as a consequence of shedding, as calreticulin was found by ELISA in the cell supernatants of stimulated cells. Antibodies raised against C1q-R and calreticulin demonstrated a high degree of immunological cross-reactivity for purified calreticulin as determined by dot blot analysis. Western blots of neutrophil subcellular fractions located calreticulin in both the cytosol and cell membrane fractions; C1q-R was largely confined to the cell membrane. Calreticulin and C1q-R both bind to C1q and mannan-binding protein. Therefore, calreticulin may be shed on cell activation and may be associated with the cell membrane, where it can potentially interact with C1q and serum lectins. The implications of this are discussed.

Calcium-Binding Proteins↗
PubMed1994

An apparently common mechanism of generating antibody diversity: length variation of the VL-JL junction.

The joining of various V, (D) and J gene segments during DNA rearrangement of the antigen receptor genes is one of the principle mechanisms responsible for the generation of antibody diversity. In the absence of N-segment variation, the structures of the coding joints formed during light chain rearrangement are thought to be less complex than their heavy chain counterparts. Consequently, the joining of the VL and JL gene segments during recombination account for all of the junctional diversity seen within the third complementarity determining region (CDR3). We generated kappa light chain transcripts from human fetal liver and peripheral blood lymphocytes and found that approximately one third exhibit a variation in the length of CDR3-independent of the JK gene segment utilized. Nucleotide sequence analysis reveals that many of the nucleotides at the VK-JK joint resulting in length variation of CDR3 are directly encoded by the germline VK and JK gene segments used in these transcripts. However, nearly 20% of the transcripts contain N-segment additions consistent with TdT-like activity. These observations suggest that TdT or an analogous enzyme must be active in a significant percentage of human B-lymphocytes during light chain rearrangement. Length variation in light chain CDR3 expands the potential repertoire and thus contributes an additional means of generating diversity in the antibody molecule.

Amino Acid Sequence↗
PubMed1994

The cross-reactive idiotopes recognized by the monoclonal antibodies 9G4 and LC1 are located in framework region 1 of two non-overlapping subsets of human VH4 family encoded antibodies.

The monoclonal anti-idiotopic antibodies LC1 and 9G4 bind two non-overlapping sets of VH4 encoded antibodies. 9G4 exclusively binds VH4-21 encoded antibodies, while LC1 binds antibodies derived from VH4 family gene segments V71-2, V71-4, VH4-18, VH72-1 and V2-1. The VH4-21 gene segment is utilized by most cold agglutinin (CA) antibodies with I/i specificity, while antibodies encoded by other VH4 gene segments are associated not with CA disease, but primarily with rheumatoid-factor (RF) activity. We previously determined that the idiotope to which 9G4 binds in VH4-21-derived antibodies is located in framework region 1 (FR1). In the present study, by using mutational analysis involving individual framework- and complementarity-determining region exchanges between VH4-21- and V71-2-encoded antibodies, we have found that the idiotope to which LC1 binds in V71-2-derived antibodies also maps to FR1. The LC1 idiotope is heavy (H)-chain associated, but requires pairing with a light (L) chain for LC1 binding. Recombinant antibodies composed of a variety of kappa (kappa) and lambda (lambda) L chains paired with either a V71-2 or VH4-21 chain were produced in the baculovirus expression system. LC1 bound all of the kappa-containing antibodies but did not bind the V71-2-encoded H chain alone nor to the two lambda-containing antibodies. This experiment demonstrates that not all light chains exert equivalent influence on the conformation of the H-chain idiotope. These results indicate that the FR1 of VH4-encoded antibodies is immunogenic and suggest a physiological role of FR1 during an immune response.

Amino Acid Sequence↗
PubMed1993

TAP1 alleles in insulin-dependent diabetes mellitus: a newly defined centromeric boundary of disease susceptibility.

It has been previously demonstrated that individuals with certain DR alleles have an increased relative risk of developing insulin-dependent diabetes mellitus (IDDM). The disease association is even stronger with certain DQ alleles but there is little association with DP providing a boundary of disease association to the 430 kb between DQ and DP. The recently described TAP (transporter associated with antigen processing) genes have been mapped approximately midway between DP and DQ. Therefore, it was of interest to determine if any TAP alleles were associated with IDDM. In addition to the alleles of TAP1 that have been described, others were identified during this study. Diabetics and normal controls were screened for TAP1 using single-stranded conformational polymorphism and relative risk was determined. In the same population group we have studied extensively in the past, we found a higher association of a TAP1 allele with IDDM than with any single HLA-DP allele but the risk was lower than with HLA-DQB1*0302. These data provide new limits for IDDM susceptibility to the 190-kb interval between TAP1 and HLA-DQB1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Molecular characterization of monoclonal CRIA-positive anti-arsonate antibodies derived from idiotype-negative mice bearing a light chain polymorphism.

We have elicited anti-arsonate antibodies bearing the major cross-reactive idiotype (CRIA) in a double congenic idiotype-negative strain (C.C58.AL-20) bearing a light chain polymorphism that has previously been shown serologically not to complement idiotype-positive heavy chains. Using the idiotype cascade (Ab1-->Ab2-->Ab3-->-->Ab1'), CRIA-positive antibodies were raised and monoclonal antibodies were isolated and characterized serologically and by nucleotide sequence analysis. Two types of idiotype-positive anti-arsonate antibodies were generated in the C.C58.AL-20 strain. One group of hybridomas used the canonical VH1.8 heavy chain gene segment with V kappa 10 variant light chains. A second group used a VHGAM3.8 heavy chain with V kappa 10 variant light chains. This latter heavy-light pairing has been observed in CRIA-like responses previously in BALB/c mice after idiotypic manipulation (or rarely after antigen alone). These studies demonstrate the plasticity of the immune response when manipulated with idiotype reagents as well as its structural variability. Additionally, they provide important insights into the potentials of idiotype vaccines.

Amino Acid Sequence↗