Biomedical subjects

K N Potter

Publications and source records attributed to K N Potter.

At least 19 recordsLinked to original sources

PubMed2000

Evidence that human immunoglobulin M rheumatoid factors can Be derived from the natural autoantibody pool and undergo an antigen driven immune response in which somatically mutated rheumatoid factors have lower affinities for immunoglobulin G Fc than their germline counterparts.

The question of whether immunoglobulin (Ig)M rheumatoid factors (RF) arise as the result of an abnormal expansion of already existing clones producing natural autoantibodies or emerge as new clones that are somatically mutated owing to an antigen driven immune response has never been conclusively answered. In this study, an inhibition ELISA was utilized to measure the affinities of recombinant antibodies using VH segments reverted back to their closest germline counterparts (germline revertants). In all cases, the somatically mutated parental RFs had a decreased affinity for immunoglobulin (Ig)G Fc compared to the germline revertant, indicating that the antibodies in the germline configuration had the higher affinities. This demonstrates that somatic mutation is not a prerequisite to generate disease associated antibodies. The presence of mutations in the parental IgM RFS suggests that these cells had been involved in a germinal centre reaction. As the germinal centre is the conventional site of the acquisition of mutations during an antigen driven response, these data suggest a role for germinal centres in the generation of the antibody diversity in addition to the selection of higher affinity antibodies. Assuming that only antigen selected cells survive deletion, these data support the hypothesis that IgM RFS can be derived from the natural autoantibody repertoire and result from an antigen driven response. Mechanisms controlling the survival of B cells based on the affinity/avidity of the immunoglobulin receptor are shown to be functional in patients with rheumatoid arthritis.

Amino Acid Sequence↗

PubMed1999

Learning-related fos-like immunoreactivity in the chick brain: time-course and co-localization with GABA and parvalbumin.

Previous work has shown that, after domestic chicks have learned the characteristics of an object (visual imprinting), there is a learning-related increase in the numerical density of Fos-immunopositive neurons in the intermediate and medial part of the hyperstriatum ventrale, a forebrain region that is a site of recognition memory for the imprinted object. The present study describes the time-course of this effect and has used double-labelling immunocytochemistry to identify neuronal types in which the effect occurs. Chicks were trained by exposure for 1 h to an imprinting (training) stimulus and then given a preference test to determine the strength of imprinting (i.e. of learning). Strongly imprinted chicks were killed 2, 2.5, 3, 3.5 or 4 h (12 chicks in each group) after the start of training and a further group of 12 chicks remained untrained. Sections from the chicks' brains were stained for Fos-like immunoreactivity, and the numerical density of Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale was counted. Relative to untrained chicks, there was a 60% increase in the number of Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale 2 h after the start of training (P = 0.02), but not at any other time. Sections from 10 trained chicks, two killed at each of the above times after training, and from two untrained chicks were stained with anti-Fos antibody as before and also with an antibody against GABA. Approximately 95% of the Fos-positive neurons in the intermediate and medial part of the hyperstriatum ventrale were also immunopositive for GABA. In neurons immunopositive for GABA, there were significantly (P = 0.02) more Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale 2 h after the start of training than in untrained chicks. Five chicks killed 2 h after training and five untrained chicks yielded sections for the next experiment; sections were double labelled for (i) Fos and (ii) either Calbindin-D28k or parvalbumin. Training gave rise to a significant (P = 0.017) increase in numerical density of Fos-positive nuclei of neurons that were immunonegative for Calbindin-D28k. This increase occurred in neurons that were immunopositive for parvalbumin. The use of alternative antibodies for GABA, Calbindin-D28k and parvalbumin in trained and untrained chicks confirmed the double-staining pattern observed in the quantitative experiments. The results demonstrate that the learning-related increase in Fos-like immunoreactivity following training is transitory and have localized the increase to a population of neurons immunopositive for GABA and parvalbumin, but not Calbindin-D28k.

Animals↗

PubMed1999

Molecular characterization of the VH1-specific variable region determinants recognized by anti-idiotypic monoclonal antibodies G6 and G8.

Mutational analysis was used to determine the structural basis for the binding of the murine anti-idiotypic (anti-Id) monoclonal antibodies (MoAb) G6 and G8 to VH1-encoded antibodies. The MoAb G6 binds a cross-reactive idiotope present on the heavy (H) chains of immunoglobulins (Ig) encoded by 51p1-related gene segments, but not those encoded by hv1263-related gene segments. Gene segments 51p1 (DP-10) and hv1263 differ by only four amino acids; three in complementarity-determining region 2 (CDR2), and one in framework region 3 (FR3). The MoAb G8 also binds 51p1-related sequences, although it is undetermined whether G8 can bind hv1263-related sequences. In order to localise the Ids recognized by MoAbs G6 and G8 on 51p1-encoded antibodies, recombinant antibodies containing H-chain mutants were expressed in insect cells. Idiotypic analysis on the expressed recombinant proteins definitively localised the reactivity in each molecule. These studies should be important in the structural appreciation for critical serological reagents.

Amino Acid Sequence↗

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

Anti-idiotypic antibody D12 and superantigen SPA both interact with human VH3-encoded antibodies on the external face of the heavy chain involving FR1, CDR2 and FR3.

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V(H)3 encoded antibodies. We used mutational analysis to determine the subregions of a V(H)3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FRI, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V(H)3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.

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

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

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↗

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

Molecular characterization of a cross-reactive idiotope on human immunoglobulins utilizing the VH4-21 gene segment.

The anti-idiotypic (anti-Id) antibody (Ab) 9G4 binds a cross-reactive idiotope (CRI) present in a select group of human autoantibodies. This Id has been localized to the portion of immunoglobulin (Ig) heavy (H) chains encoded by the VH4-21 gene segment, a member of the human VH4 family. This gene segment is utilized by essentially all cold agglutinin (CA) Abs with I/i specificity isolated from patients with CA disease stemming from chronic lymphoproliferative disorders. In this study, mutational analysis of a CA has been used to determine the structural basis for 9G4 binding to Abs utilizing the VH4-21 gene segment. Recombinant CA H chain mutants were produced and their 9G4 reactivity determined. Mutants were generated by exchanging VH4-21 sequences in the FR1, CDR1, and CDR2 with corresponding sequences from a closely related gene segment V71-2, a VH4 family member that is associated neither with Abs having CA activity nor with Abs that react with 9G4. The results indicate that the motif AVY at amino acid positions 23-25 in FR1 defines the 9G4 idiotope. Reaction of these recombinant Abs with a polyclonal rabbit anti-CA antiserum absorbed to render it specific for a CA CRI also maps predominantly to FR1. These findings indicate that the solvent-exposed FR1 plays an important role in eliciting an immune response to Igs.

Agglutinins↗

PubMed1993

Complementarity-determining region 2 is implicated in the binding of staphylococcal protein A to human immunoglobulin VHIII variable regions.

Staphylococcal protein A (SPA) has two distinct binding sites on human immunoglobulins. In addition to binding to the Fc region of most IgG molecules, an "alternative" binding site has been localized to the Fab region of human immunoglobulins encoded by heavy chain variable gene segments belonging to the VHIII family. Comparison of amino acid sequences of closely related SPA-binding and -non-binding proteins suggested that VHIII-specific residues in the second complementarity-determining region (CDR2) were likely responsible for SPA binding activity. Site-directed mutagenesis of a single amino acid residue in CDR2 converted an IgM rheumatoid factor which did not bind SPA to an SPA binder. These findings, therefore, locate a critical site involved in SPA binding to the CDR2 of human immunoglobulins encoded by VHIII family gene segments.

Amino Acid Sequence↗

PubMed1993

Antibody production in the baculovirus expression system.

The proven ability of insect cells to produce murine and human antibodies renders the baculovirus system amenable to the synthesis of both naturally occurring antibodies and designed mutants. Study of these antibodies should provide a basis for rational antibody design useful in human therapy.

Animals↗

PubMed1992

Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes.

Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria.

DNA Probes↗

PubMed1986

Methylation blockage and other improvements to a comprehensive DNA analysis program.

A comprehensive DNA analysis computer program was described in the second special issue of Nucleic Acids Research on the applications of computers to research on nucleic acids by Stone and Potter (1). Criteria used in designing the program were user friendliness, ability to handle large DNA sequences, low storage requirement, migratability to other computers and comprehensive analysis capability. The program has been used extensively in an industrial-research environment. This paper talks about improvements to that program. These improvements include testing for methylation blockage of restriction enzyme recognition sites, homology analysis, RNA folding analysis, integration of a large DNA database (GenBank), a site specific mutagenesis analysis, a protein database and protein searching programs. The original design of the DNA analysis program using a command executive from which any analytical programs can be called, has proven to be extremely versatile in integrating both developed and outside programs to the file management system employed.

Base Sequence↗

PubMed1984

A DNA analysis program designed for computer novices working in an industrial-research environment.

A program designed for DNA sequence analysis by people with no computer background or experience is described. The criteria used in the program design were (i) user friendliness, (ii) ability to handle large DNA files (greater than 10 kilobases), (iii) low storage requirements so that it could be put on a personal computer (IBM), (iv) written in a widely used language (FORTRAN 77) and (v) ability to handle all of the needed tasks for DNA analysis. The program consists of an executive that controls a set of modular subprograms. A file management system using 'SEQ' and 'SITE' files is also in place. SEQ files contain long DNA nucleotide sequences (e.g. restriction enzyme sites). Related SEQ and SITE files are grouped into categories. A SITE category can include related sequences a user wishes to search for in a longer SEQ sequence from a designated category. For example, all of the restriction enzyme recognition sites can group into a single category called 'Enzymes' and can be searched for in a SEQ sequence which has been placed in the SEQ file category 'Plasmids'. Colour graphics is also used as a visual aid.

Base Sequence↗

PubMed1978

Modification of Trichoplusia ni nuclear polyhedrosis virus passaged in vivo.

A designated strain of plaque-purified Trichoplusia ni nuclear polyhedrosis virus (NPV) was used to initiate a serial passage series in vivo. The virulence of polyhedra inclusion bodies (PIB) and the distribution of MP (many PIB) and FP (few PIB) strains in hemolymph were monitored. When virus was passaged per os as PIB, there was no major change in virulence after 15 passes in insects. Plaque analysis of hemolymph from the infected insects indicated that there was no selection for the FP strain of virus. There was little overall change in the infectivity of nonoccluded virus (NOV) after 16 passes by injection into T. ni larvae; however, plaque analysis of the hemolymph from the infected insects indicated progressive selection of FP virus. It is postulated that when PIB are the vehicles of virus transmission, there is no progressive accumulation of noninfectious FP PIB; however, when NOV are the vehicles of virus transmission, FP NOV production displaces MP virus synthesis. This may result from a growth advantage of the FP NOV over the MP NOV as shown in vitro and from the noninfectious nature of FP PIB.

Animals↗

PubMed1976

Strain selection during serial passage of Trichoplusia in nuclear polyhedrosis virus.

Two strains of a nuclear polyhedrosis virus (NPV) of Trichoplusia ni were isolated on the basis of plaque morphology. They are designated as MP (having greater than 30 polyhedra per nucleus) and FP (having fewer than 10 polyhedra per nucleus). Serial, undiluted passage of plaque, purified MP nonoccluded. Virus (NOV) in tissue culture led to the production of the FP phenotype detectable at passage 9. With continued serial, undiluted passage, FP became the predominant strain. Comparative growth curves showed that FP NOV are released faster than MP NOV. MP morphology was not observed after 14 serial, undiluted passages of plaque-purified FP. By the plaque neutralization assay, NOV from both strains of virus was neutralized by the homologus and heterologous antisera. The FP phenotype was observed when FP virus was grown in culture at 17, 22, and 27 C. Hence, the FP phenotype was not considered to be the result of temperature-inhibited crystallization of polyhedrin under standard tissue culture conditions. The NOV of both strains killed insects when injected directly into the hemocoele of T. ni larvae. Only MP inclusion bodies were virulent per os. The FP inclusion bodies fed to cabbage looper larvae did not kill, and no infectious agent could be detected in the hemolymph. Electron micrographs of MP polyhedra showed bundles of nucleocapsids of normal length within the polyhedra, whereas FP polyhedra contained heterogeneous, electron-dense material, which could account for their lack of pathogenicity.

Animals↗