Structure-function studies of anti-3-fucosyllactosamine (Le(x)) and galactosylgloboside antibodies.

We are studying murine mAbs against two carbohydrate epitopes, 3-fucosyllactosamine (Le(x), CD15) and galactosylgloboside. The VH domains of both panels of Ab are encoded by VH441, a member of the X24 family of Ig genes. To evaluate the contribution of the heavy chain CDR3 to the affinity of the anti-3-fucosyllactosamine Ab, CDR3-H of PMN6, a low affinity Ab, was replaced by the CDR3 of PM81, a higher affinity Ab. The affinity of the chimeric 6/81 Ab was increased when the heavy chain was paired with the PM81 light chain, but not when paired with another light chain (M5), which differs from PM81 light chain by three amino acids. To evaluate the contribution of somatic mutations to the binding of GalGb4, the 3A9 VH sequence, which contains three amino acid substitutions, was replaced by a germ-line sequence encoded by either VH441 or VHX24. The chimeric Ab, 441/3A9 and X24/3A9, bound Ag as well as the wild-type 3A9 Ab. Computer models of the Fv fragments of PM81 and 3A9 were compared with the crystal structure of the Fv fragment of J539, a galactan-binding myeloma protein that is encoded by the same VH and VK genes as 3A9. The surfaces of 3A9 and J539 have shallow pockets that are potential Ag-binding sites. Replacement of CDR3-H Tyr99, which is a prominent component of the pocket, by Ala abolished the binding of Ag. In contrast, the Fv surface of PM81 contains a large cleft rather than a pocket. These models indicate how the same VH gene segment can be used to encode Abs that exhibit different specificities.

Polymorphism of human immunoglobulin VH4 germ-line genes.

The human immunoglobulin VH4 gene family is thought to contain approximately 10 germ-line genes and to exhibit little polymorphism. We report here an analysis of VH4 germ-line genes that were amplified from DNA of two unrelated individuals. Ten unique (non-repetitive) sequences were obtained from individual A and 11 from individual B. Nine of these sequences represent new germ-line genes, and 8/9 exhibit only 89%-96% similarity to genes identified previously. Subsets of VH4 genes displayed distinctive nucleotide motifs that account for most of the differences between them. This observation suggests that diversity in the VH4 gene family arose from the acquisition of blocks of nucleotides, rather than by accumulation of point mutations. These nucleotide blocks could have been acquired by gene conversion or by homologous recombination. All of the VH4 genes have a potential N-linked glycosylation site at Asn 60, and some genes encode a second site at Asn 52. The VH4 gene family is larger and more polymorphic than appreciated previously. Immunoglobulin gene polymorphism may make a significant contribution to hereditary variations in the immune response and to the genetic predisposition to autoimmune diseases.

Heavy and light chain sequences of four monoclonal antibodies that bind galactosylgloboside (GalGb4).

We recently described IgM monoclonal antibodies directed against the glycospingolipid galactosylgloboside (GalGb4; Marcus, M. D. et al., Arch. Biochem. Biophys, 1988.262: 620). We now present the nucleotide and deduced amino acid sequences of the heavy and light chains of these antibodies. The antibodies were generated in a single fusion, their heavy as well as their light chains are almost identical, and they appear to be clonally related. The light chains were encoded by J kappa 5 and a V kappa gene belonging to the Ox1 family, but they are only 93% homologous to the most closely related germ-line gene, and they are probably encoded by a germ-line gene that has not yet been identified. The heavy chains were all encoded by VH441 and JH2, and have identical N segments. The VH441 germ-line gene encodes a potential glycosylation site at Asn58 in the complementarity-determining region 2. This site, which has been retained in all VH441-encoded monoclonal antibodies sequenced previously, was mutated out by a single base change in all four anti-GalGb4 antibodies.

Binding of actinomycin D to DNA: evidence for a nonclassical high-affinity binding mode that does not require GpC sites.

We have employed a combination of temperature-dependent UV absorption spectroscopy, circular dichroism, and batch calorimetry to characterize the binding of actinomycin D to a series of oligomeric DNA duplexes. We find the duplex [d(CGTCGACG)]2 to be unique in its ability to bind actinomycin D strongly despite the absence of a classic GpC site. We present evidence that this non-GpC-containing duplex binds two actinomycin D molecules in an apparently cooperative manner to form a complex that exhibits aberrant spectroscopic and calorimetric behavior. We propose that these observations are consistent with actinomycin D exhibiting a high-affinity, sequence-dependent DNA-binding mode distinct from its classic binding to isolated GpC sites.

Enthalpy-entropy compensations in drug-DNA binding studies.

We present a comparative study of calorimetrically derived thermodynamic profiles for the binding of a series of drugs with selected DNA host duplexes. We use these data to demonstrate that comparisons between complete thermodynamic profiles (delta G zero, delta H zero, delta S zero, delta Cp) are required before drug binding can be used as a probe of DNA conformation, since enthalpy-entropy compensations can cause two drug-DNA binding events to exhibit similar binding free energies (delta G zero) despite being driven by entirely different thermodynamic forces (delta H zero, delta S zero). In this work, we employ a combination of spectroscopic and calorimetric techniques to characterize thermodynamically the DNA binding of netropsin and distamycin (two minor groove-directed ligands), ethidium (an intercalator), and daunomycin (a combined intercalator/groove binder). Our free energy data (delta G zero) show that each drug exhibits similar binding affinities at 25 degrees C for the alternating copolymer duplex poly[d(A-T)].poly[d(A-T)] and for the homopolymer duplex poly(dA).poly(dT). However, our calorimetric measurements reveal that the nature of the thermodynamic forces (delta H zero, delta S zero) that drive drug binding to these two host duplexes at 25 degrees C are entirely different, despite similar binding free energies (delta G zero) and similar salt dependencies (lnK/ln[Na+]). Specifically, the 25 degrees C binding of all four drugs to the alternating copolymer poly[d(A-T)].poly[d(A-T)] is overwhelmingly enthalpy driven, whereas the corresponding binding of each drug to the homopolymer duplex poly(dA).poly(dT) is overwhelmingly entropy driven. Thus, the similar binding free energies (delta G zero) we measure for complexation of each drug with poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) result from compensating changes in the enthalpy and entropy terms. Comparison with the thermodynamic profiles for the complexation of these drug molecules to other DNA host duplexes at 25 degrees C reveals that the binding of each is strongly enthalpy driven, except when the poly(dA).poly(dT) homopolymer serves as the host duplex. This comparison allows us to conclude that poly[d(A-T)].poly[d(A-T)] behaves thermodynamically as the more "normal" host duplex toward drug binding, whereas the entropy-driven binding to the poly(dA).poly(dT) duplex represents "aberrant" behavior. Furthermore, since each of the four drugs exhibits different modes of DNA binding, we conclude that the observed entropy-driven behavior for binding to poly(dA).poly(dT) reflects an intrinsic property of the homopolymer duplex that is perturbed in a common manner upon ligation rather than a common property of all four binding ligands. To rationalize the large positive entropy changes that drive drug complexation with poly(dA).poly(dT) duplex, we propose a model that emphasizes binding-induced perturbations of the more highly hydrated, altered B conformation of the homopolymer. Our results suggest that an aberrant thermodynamic binding profile may reflect an unusual DNA conformation in the host duplex. However, before such a conclusion can be reached, complete thermodynamic binding profiles must be examined, since enthalpy-entropy compensations can cause two binding events to exhibit similar binding constants even when they are driven by very different thermodynamic forces.

Evaluation of the phase contrast microscopy method for the detection of fibrous and other elongated mineral particulates by comparison with a STEM technique.

The USPHS/NIOSH Membrane Filter Method is used to monitor for asbestos in occupational and mining atmospheres, and employs the phase-contrast optical microscope (PCM) that under optimum conditions has a resolution of approximately 0.25 micron. While amphibole cleavage fragments are usually visible by PCM, asbestos fibers (such as amosite and chrysotile) have finer widths that may render them invisible by PCM. In this study, personal air-monitoring filters containing chrysotile, amosite and amphibole cleavage fragments from various sources have been analyzed by PCM in accordance with the USPHS/NIOSH Method and scanning transmission electron microscopy (STEM) to assess the effectiveness of the PCM technique. Each STEM specimen was prepared using a direct-transfer technique to ensure that particle size distribution and concentration were not altered. STEM results for chrysotile samples are highly variable, with 9% to 81% of regulatory particles having widths smaller than 0.25 micron--the resolution of the optical microscope. Amosite samples have 27% to 38% of regulatory particles with widths below microscope resolution, indicating that routine particle counts by PCM on these samples would underestimate true fiber content by approximately one-third. All amphibole cleavage fragment samples had regulatory particles that would be observed by PCM. Multiplication factors have been suggested for application to routine counts by PCM to more accurately assess true particle content for mineral particulates on personal air-monitoring filters.

Thermodynamics of drug-DNA interactions.

Batch calorimetry, differential scanning calorimetry (DSC), uv/vis absorption spectroscopy, fluorescence spectroscopy, and circular dichroism (CD), have been used to detect, monitor, and thermodynamically characterize the binding of daunomycin, dipyrandenium, dipyrandium, and netropsin to poly d(AT) and actinomycin D to salmon testes (ST) DNA. The following thermodynamic binding profiles have been obtained. (table; see text) All the poly d(AT) binding studies were done at 25 degrees C while actinomycin binding to ST DNA was performed at 1 degree C to enhance drug solubility. These thermodynamic parameters are interpreted in terms of specific interactions that have been proposed as part of models for the binding of each drug.

Calorimetric and spectroscopic investigation of drug--DNA interactions: II. Dipyrandium binding to poly d(AT).

We report the first calorimetric investigation of steroid diamine binding to a DNA duplex. Absorption spectroscopy, batch calorimetry, and differential scanning calorimetry (DSC) have been used to detect, monitor, and thermodynamically characterize the binding of the steroid diamine, dipyrandium, to poly d(AT). The following thermodynamic data for the binding in 16 mM Na+ at 25 degrees C have been obtained: delta G degree = -6.5 kcal/mol, delta H degree = +4.2 kcal/mol, and delta S = +36 e.u. We interpret the endothermic binding enthalpy in terms of steroid-induced conformational changes in the duplex (e.g. "kinking"). The large positive entropy is interpreted in terms of binding-induced release of bound water and condensed sodium ions. The salt-dependence of the binding constant is interpreted in terms of dipyrandium site-binding involving only one of the two charged ends of the steroid. The optical and DSC curves for the unsaturated steroid-poly d(AT) complexes exhibit biphasic behavior. A comparison of the van't Hoff and the calorimetric transition enthalpies reveals that steroid binding reduces the cooperativity of the transition.