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Biomedical subjects

A M Giusti

Publications and source records attributed to A M Giusti.

46 records · Page 3Linked to original sources
PubMed1991

Deoxyribonucleic acid (DNA) analysis by restriction fragment length polymorphisms of blood and other body fluid stains subjected to contamination and environmental insults.

Deoxyribonucleic acid (DNA) restriction fragment length polymorphism (RFLP) profile results were obtained from bloodstains and other body fluid stains subjected to mixture with other body fluids, environmental insults (sunlight and temperature), different substrates (cotton, nylon, blue denim, glass, aluminum, and wood), and contaminants (gasoline, bleach, sodium hydroxide, soil, motor oil, detergent, phosphate salt, glacial acetic acid, and microorganisms). Of the samples that produced profile results, all had profiles that were consistent with those of untreated control samples.

Blood Stains↗
PubMed1990

Applying highly polymorphic variable number of tandem repeats loci genetic markers to identity testing.

The detection of alleles of variable number of tandem repeats (VNTR) loci by restriction fragment length polymorphism analysis has become an important aspect of genetic characterization for identity testing. Some VNTR loci are so polymorphic that the analysis of three to five genetic markers could potentially provide unique identity. However, the more informative a genetic marker is (i.e., high degree of polymorphisms), the better it is as an exculpatory tool. This approach currently provides the best avenue for excluding a falsely associated individual with a particular sample. When an analysis fails to exclude an individual as the source of the questioned material, a value (frequency of occurrence) should be placed on the VNTR profiles to assess weight to the inclusion in identity testing. Arbitrarily defined fixed bins were designed to accommodate quasi-continuous data and to provide a result that would not place an underestimation of the frequency of occurrence of a set of alleles attributed to an individual.

Alleles↗
PubMed1989

Significant structural and functional change of an antigen-binding site by a distant amino acid substitution: proposal of a structural mechanism.

To study the molecular basis for antibody diversity and the structural basis for antigen binding, we have characterized the loss of phosphocholine (P-Cho) binding both experimentally and computationally in U10, a somatic mutant of the antibody S107. Nucleotide sequencing of U10 shows a single base change in JH1, substituting Asp-101 with Ala, over 9 A distant from the P-Cho-binding pocket. Probing with antiidiotypic antibodies suggests local, not global, conformational changes. Computational results support a specific structural mechanism for the loss of P-Cho binding. The U10 mutation eliminates the charged interaction between Asp-101 and Arg-94, which allows the Arg-94 side chain to disrupt P-Cho binding sterically and electrostatically by folding into the P-Cho-binding site. These results specifically show the importance of the Arg-94 to Asp-101 side chain salt bridge in the heavy-chain CDR3 conformation and suggest that residues distant from the binding site play an important role in antibody diversity and inducible complementarity.

Animals↗
PubMed1987

Somatic diversification of S107 from an antiphosphocholine to an anti-DNA autoantibody is due to a single base change in its heavy chain variable region.

The S107 myeloma cell line expresses the germ-line sequence of the T15 antiphosphocholine (P-Cho) antibody, which is the major antibody made by BALB/c mice in response to P-Cho, either on a variety of bacterial polysaccharides or when attached to a protein carrier. We have previously reported that a somatic mutant of the S107 cell line produces an antibody that has lost the ability to bind P-Cho and has acquired binding for double-stranded DNA. This antibody has a substitution of an alanine for a glutamic acid at residue 35 in the heavy chain variable region. We now show that this amino acid substitution is due to a single A-C transversion, which is the only nucleotide change in the heavy and light chain variable regions. Further, it appears that this change is due to somatic mutation rather than to gene conversion.

Animals↗
PubMed1984

Rat anti-T15 monoclonal antibodies with specificity for VH- and VH-VL epitopes.

BALB/c mice immunized with phosphorylcholine (PC) produce antibodies which are predominantly of the T15 idiotype. Monoclonal anti-T15 antibodies have been generated in a number of laboratories by allogenic or syngenic immunization. Most of these mouse monoclonal antibodies react with idiotopes that are in or near the PC-binding site and require the presence of both T15 heavy and T15 light chain variable regions. By immunizing rats with T15 immunoglobulins, we have obtained monoclonal antibodies that recognize idiotopes that are not near the antigen-binding site. Four of these rat anti-T15 monoclonal antibodies react with free T15 heavy chains and with T15 heavy chains associated with irrelevant light chains while four other rat monoclonal antibodies require the presence of both T15 heavy and T15 light chains. This battery of rat anti-T15 monoclonal antibodies is useful in searching for heterogeneity within the T15 antibodies and in following the expression of the T15 heavy chain variable region in different strains of mice.

Animals↗
PubMed1983

Monoclonal antibodies reveal the structural basis of antibody diversity.

Hybridoma technology has made it possible to introduce into continuous culture normal antibody-forming cells and to obtain large amounts of the immunoglobulin produced by each of these cells. Examination of the structure of a number of monoclonal antibodies that react with a single antigen has provided new information on the structural basis of the specificity and affinity of antibodies. Comparisons of families of monoclonal antibodies derived from a single germ line gene revealed the importance of somatic mutation in generating antibody diversity. Monoclonal antibodies that react with variable regions of other monoclonals allow the further dissection and modulation of the immune response. Finally, the continued somatic instability of immunoglobulin genes in cultured antibody-forming cells makes it possible to determine the rate of somatic mutation and to generate mutant monoclonal antibodies that may be more effective serological reagents.

Amino Acid Sequence↗
PubMed1982

Somatic mutation in a cultured mouse myeloma cell affects antigen binding.

The S107 mouse myeloma cell line synthesizes an IgA antibody that binds the hapten phosphocholine and is similar if not identical in its heavy and light chain variable region sequence to the predominant antibody produced by BALB/c mice in response to immunization with phosphocholine. This cell line frequently and spontaneously generates somatic variants producing immunoglobulins with decreased ability to bind antigen. One such variant, S 107.U1, is described here. This variant has a decreased ability to bind phosphocholine when it is attached to a carrier, although its affinity for free hapten is the same as that of the parent. This decrease in antigen binding is associated with a single amino acid substitution at the fifth residue in the JH segment.

Amino Acid Sequence↗
PubMed1982

Single amino acid substitution altering antigen-binding specificity.

S107, a phosphocholine-binding myeloma protein, has been cloned in soft agar, and an antigen-binding variant has been isolated and characterized. The variant does not bind phosphocholine attached to carrier or as free hapten in solution but does retain antigenic determinants (idiotypes) of the parent. Chain recombination experiments suggest that the defect in binding is entirely in the heavy chain. Amino acid sequence analysis showed a single substitution--glutamic acid to alanine at position 35--in the first hypervariable or complementarity-determining region. In terms of the three-dimensional model of the phosphocholine-binding site, glutamic acid-35 provides a hydrogen bond to tyrosine-94 of the light chain that appears to be critical for stability of this portion of the binding site. The removal of this bond and the presence of the smaller alanine side chain is thus consistent with the loss in binding activity. These results suggest that small numbers of substitutions in antibodies, such as those presumably introduced by somatic mutation, may in some situations be effective in altering antigen-binding specificity.

Amino Acid Sequence↗