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J D Capra

Publications and source records attributed to J D Capra.

At least 37 records · Page 2Linked to original sources
PubMed1998

4-Aminoquinoline antimalarials enhance UV-B induced c-jun transcriptional activation.

Previous work has documented that the earliest observable response in mammalian cells following ultraviolet (UV) irradiation is the activation of plasma membrane-associated Src tyrosine kinases. These molecules then trigger a signalling cascade that results in activation of the transcription factor AP-1 which subsequently transactivates the early immediate genes including c-jun. This pathway has been postulated to play a protective role against UV damage. As aminoquinoline antimalarials such as chloroquine are known to downregulate several photoinduced cutaneous disorders including LE-specific skin disease, we asked whether chloroquine might be capable of modulating this early limb of the UV light response. A431 cells (a human epidermal keratinocyte cell line) that had been transfected with a c-jun luciferase reporter gene construct were then treated with physiologically relevant concentrations of chloroquine followed by exposure to 0-125 J/m2 of UV-B from a bank of unfiltered FS20 lamps. Chloroquine pretreatment resulted in a dose-dependent increase in luciferase activity in permanently transfected A431 cells (luciferase activity was increased by 45% at 2.5 x 10(-5) M chloroquine and 125 J/m2 of UV-B). Hydroxychloroquine pretreatment also resulted in an increase in luciferase activity. Primaquine, an 8-aminoquinoline, did not influence the UV-B induced c-jun activity. Furthermore, chloroquine did not have a similar impact on HSP-70 gene activity during heat shock. These studies suggest that the beneficial effect of the 4-aminoquinoline antimalarials in various photodermatoses including cutaneous LE might result in part from the capacity of these drugs to enhance the protective early limb of the UV response.

Aminoquinolines↗
PubMed1997

NonO enhances the association of many DNA-binding proteins to their targets.

NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation.

Antigens, Nuclear↗
PubMed1997

Variable region gene segment utilization in rhesus monkey hybridomas producing human red blood cell-specific antibodies: predominance of the VH4 family but not VH4-21 (V4-34).

Structural analyses of human immunoglobulin gene segments from monoclonal cell lines provide valuable information regarding the antibody repertoire. This information, in conjunction with a nearly complete knowledge of the human immunoglobulin germline repertoire, now allows further investigation into the underlying molecular basis responsible for some of the observed biases found in the expressed repertoire. One human heavy chain variable region gene segment, V4-34 (VH4-21), is one of the most prevalent gene segments in the expressed repertoire. The overwhelming presence of the V4-34 gene segment suggests that it may play an important role in immune responses. However, there is currently little information regarding its presence and potential importance in nonhuman primates. In order to determine if this gene segment is used by lower primates in a similar manner we determined the molecular structure of the variable region gene segments that are expressed by macaque monoclonal heterohybridomas that are specific for human red blood cell antigens. Eleven of the 12 hybridomas are derived from Rhesus monkeys (Macaca mulatta) and one is from a cynomologous monkey (Macaca fascicularis), all of which have been immunized with human red blood cells. The predominance of a VH4-like family and the specific absence of a VH4-21 equivalent led us to further characterize the macaque VH4 gene family at the germline level. Therefore, germline gene segments from the macaque equivalent to the human VH4 gene family are also described.

Animals↗
PubMed1997

Induction of somatic mutation in a human B cell line in vitro.

Both the B cell-surface trigger(s) and the intracellular molecular mechanism(s) of somatic hypermutation in immunoglobulin (Ig) variable region genes remain unknown, partly because of the lack of a simple and reproducible in vitro model. Here, we show that upon surface immunoglobulin cross-linking followed by co-culture with activated cloned T cells, the Burkitt's lymphoma cell line BL2 is induced to mutate its IgV(H) gene. Repeated activation of BL2 cells increased the frequency of mutation. The in vitro-induced mutations, which do not affect the IgM constant region, are point mutations distributed over the entire V(H)DJ(H) gene segment and do not show evidence of antigen-driven selection.

Amino Acid Sequence↗
PubMed1997

Staphylococcal protein A binding to VH3 encoded immunoglobulins.

Staphylococcal protein A (SPA) is a B-cell superantigen which binds specifically to the variable region of human VH3 encoded antibodies. We undertook to identify the VH3 regions involved in the interaction with SPA by producing mutant antibodies in the baculovirus expression system. We had previously shown that a single amino acid change at position 57 in the CDR2 of a human SPA nonbinding VH3 encoded rheumatoid factor converted it to an SPA binder, implicating CDR2 in SPA binding. When regions of the mutated binder were exchanged with those from a mouse nonbinding antibody, the pattern of SPA binding indicated that residues in FR1, CDR2 and FR3 are involved in the interaction between VH3 encoded antibodies and SPA. In addition, all three regions are simultaneously required for SPA binding to occur. When any one of the three regions was altered, SPA binding was severely disrupted.

Amino Acid Sequence↗
PubMed1996

Staphylococcal protein A simultaneously interacts with framework region 1, complementarity-determining region 2, and framework region 3 on human VH3-encoded Igs.

Staphylococcal protein A (SPA) is a B cell superantigen that binds to human VH3-encoded Igs independently of the D- and JH-encoded regions and light chain sequences. The SPA-binding structure formed by VH3-encoded Igs remains controversial. We localized the regions in a VH3-encoded Ab required for SPA binding by producing mutant Abs in the baculovirus expression system in which regions of a human-derived Ab known to bind SPA were exchanged with those from a mouse Ab of the J558 family, a family not associated with SPA binding. The pattern of SPA binding indicates not only that residues in FR1, CDR2, and FR3 are involved but also that the three regions are required to interact simultaneously with SPA for binding to occur. When any one of the three regions was replaced with the corresponding region from the nonbinding Ab, SPA binding was severely disrupted. These data indicate that SPA requires simultaneous interaction with three distinct regions of a VH3 structure, which together in three-dimensional space form an extended solvent-exposed surface. These studies more precisely define the genetic requirements for VH3-encoded Ig binding to SPA.

Animals↗
PubMed1996

Calreticulin binds hYRNA and the 52-kDa polypeptide component of the Ro/SS-A ribonucleoprotein autoantigen.

Calreticulin (CR) is a multifunctional, calcium-binding protein that has recently been shown to bind to and promote the replication of the rubella virus genome in mammalian cells. While CR is now widely recognized as a new human autoantigen, the relationship between CR and the Ro/SS-A ribonucleo-protein (RNP) autoantigen has been somewhat controversial. In this work, we demonstrate that unphosphorylated human rCR binds specifically and distinctly to in vitro transcribed forms of hYRNA, the RNA backbone of the Ro/SS-A RNP particle. This interaction appears to be mediated by binding through the N- and C-terminal domains of CR, but not by the central proline-rich domain. Furthermore, our studies indicate that CR can facilitate the binding of the 60-kDa polypeptide component of the Ro/SS-A RNP (Ro60) to hYRNA. In addition, CR and the 52-kDa Ro/SS-A polypeptide (Ro52) appear to be capable of interacting through direct protein-protein binding. These studies confirm that CR is an hYRNA-binding protein, and provide for the first time a molecular mechanism by which Ro52 can be linked physically to hYRNA. Through these molecular interactions and its known functional role as a chaperone, it is suggested that CR plays a supportive role in the formation of the Ro/SS-A RNP complex. The capacity of CR to interact with RNA viruses such as rubella provides an additional argument for an infectious trigger for autoantibody production against self RNP particles such as Ro/SS-A.

Autoantigens↗
PubMed1996

Localization of the binding site for the monocyte immunoglobulin (Ig) A-Fc receptor (CD89) to the domain boundary between Calpha2 and Calpha3 in human IgA1.

Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA. Indeed, since the C alpha 2 N-linked glycosylation site lies near the Fab-distal pole of C alpha 2, the inability of a mutant IgA1 lacking C alpha 2 N-glycosylation to bind its cognate receptor suggested that the monocyte Fc alpha receptor (mFcalphaR) recognizes IgA at a hinge-distal site encompassing the boundary between the C alpha 2 and C alpha 3 domains. In this report, we utilize both domain-swapped IgA/IgG and point-mutated IgA chimeras to verify the above hypothesis. Using an antigen-specific rosetting assay and a mFc alpha R-expressing cell line, we show that (a) C alpha 2 and C alpha 3 together are necessary and sufficient for binding; (b) neither the IgA hinge nor the tailpiece is necessary for binding; (c) mutations away from the interdomain boundary do not affect binding; and (d) mutations located near the three-dimensional boundary between C alpha 2 and C alpha 3 completely disrupt binding. Taken together, these results localize the mFc alpha R recognition site on IgA to the boundary region between the second and third constant domains--a site analogous to that recognized by Staphylococcus aureus protein A on IgG. The use of this hinge-distal site is, to date, unique among Fc receptors of the Ig superfamily.

Amino Acid Sequence↗
PubMed1996

The I binding specificity of human VH 4-34 (VH 4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3.

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.

Agglutinins↗
PubMed1996

Ku is a general inhibitor of DNA-protein complex formation and transcription.

Ku is a ubiquitous and abundant DNA binding protein. Recently, it has been shown that Ku plays a crucial role in double stranded-DNA (dsDNA) break repair such as occurs during the V(D)J recombination of Ig genes. Ku has also been found to provide DNA binding activity to the catalytic domain of DNA-PK which is known to phosphorylate several transcription factors, suggesting that Ku is a multifunctional protein that participates as a component of several functional DNA-protein complexes. Here, we examined the interaction of Ku with several DNA binding proteins. Firstly, the DNA binding interaction between Ku and well-characterized transcription factors (OTF-1, Sp-1, AP-1) was analysed by EMSA. Although sequence non-specific, Ku was strongly competitive with these sequence specific transcription factors on compatible DNA elements, displacing them because of its high affinity association with DNA ends. Secondly, to determine whether this competitive effect was functionally relevant, we tested Ku in an in vitro transcription system with the adenovirus major late promoter. We found that Ku inhibited transcription from linear, but not from circular template DNA. These results suggest that Ku inhibits transcription when it is able to bind to template DNA and that the inhibition is the result of Ku displacing specific transcription factors from DNA.

Antigens, Nuclear↗
PubMed1996

The MoAb-V kappa IIIb cross-reactive idiotope on A27a (Humkv325) encoded kappa chains maps to framework region 3. off.

The monoclonal antibody MoAb-V kappa IIIb binds a cross-reactive idiotopic (CRI) determinant on light (L) chains encoded by the V kappa IIIb subgroup A27a (Humkv325) gene segment. The aim of this study was to localize the MoAb-V kappa IIIb CRI. Mutational analyses involving region exchanges between a CRI-positive V kappa IIIb chain and a CRI-negative V kappa 1 chain indicate that the MoAb-V kappa IIIb CRI is located in framework region (FR) 3 of A27a (Humkv325) encoded L chains. CRI-positive kappa chains unpaired with a heavy (H) chain are reactive with MoAb-V kappa IIIb, indicating that the CRI is located on the kappa chain alone without involvement of H chain residues. Combinatorial antibodies composed of non-parental L and H chain pairings are reactive with MoAb-V kappa IIIb only when the L chain is A27a (Humkv325) encoded. The CRI, therefore, is not readily perturbed by H chain interactions. When the FR3 from a CRI-positive kappa chain replaced the FR3 in a CRI-negative lambda chain, the determinant was no longer detectable with MoAb-V kappa IIIb. It is possible, therefore, to exchange regions between kappa chains from different families and retain the CRI structure, however the determinant is lost when placed in a more foreign background such as a lambda chain. These data more precisely define the interaction between MoAb-V kappa IIIb and its CRI, and indicate that there are limits within which antibody FRs can be shuffled and still retain their native structural features.

Amino Acid Sequence↗
PubMed1995

Characterization and genomic mapping of a novel leader peptide associated with the human VH4-21 (VH4-34) gene segment.

The human IgVH locus is located on chromosome 14, band q32 and spans approximately 1 mb. Within this locus are approximately 120 VH gene segments that are subdivided into six to seven families based on sequence homology of their coding regions. VH4-21 (VH4-34) is a member of the VH4 family, a family that contains 10 to 15 members. It is expressed in a variety of circumstances including early fetal development, the autoantibody repertoire, and in a highly restricted manner in antibodies that recognize alloantigens on the surface of human red blood cells. Most interesting, however, is the expression of this gene segment in T cells as a semi-germline transcript in conjunction with a nontraditional VH leader peptide. This nonhydrophobic leader sequence, termed "Et" for exon in T cells, has previously been shown to reside within the VH locus. Using YAC and P1 clones, we have identified two copies of this exon, both of which are located in the region of the locus that contains VH4-24 (VH4-34). Characterization of the two exons suggests that they arose by duplication, as flanking DNA is almost identical over a distance of > 5 kb. Preliminary data suggests they are both located > 20 kb upstream of VH4-21 (VH4-34).

Base Sequence↗
PubMed1995

Analysis of direct and inverted DJH rearrangements in a human Ig heavy chain transgenic minilocus.

D to JH rearrangement can occur either by a deletional or an inversional mechanism. In this study, we have analyzed deletional and inversional D to JH recombination in a human Ig heavy chain transgenic minilocus. The analysis of these events in a transgenic minilocus rather than in vivo in the human is simplified by the presence of a limited number of well defined VH, D, and JH gene segments in the minilocus. We show that in the transgenic minilocus, all D gene segments can be rearranged by deletion and virtually all can be rearranged by inversion. We also show that depending upon the D gene segment, rearrangement by deletion occurs approximately 1 to 1000 times more frequently than rearrangement by inversion. Our data suggest that in vivo, signal and coding end sequences are the major influences on the rearrangement frequency of a particular gene segment in a given orientation rather than its position within the transgenic locus. Additionally, our data indicate that intronic and recombination signal sequences are involved in the bias for deletion over inversion rather than the recombinase machinery itself.

Animals↗