Duane W. Sears

Unique Monoclonal Antibodies Define Expression of FcγRI on Macrophages and Mast Cell Lines and Demonstrate Heterogeneity Among Subcutaneous and Other Dendritic Cells

The mouse FcγRI is one of the most fundamentally important FcRs. It participates in different stages of immunity, being a low affinity receptor for T-independent IgG3 and yet a high affinity receptor for IgG2a, the product of a Th1 immune response. However, analysis of this receptor has been difficult due largely to the failure to generate specific Abs to this FcR. We have made use of the polymorphic differences between BALB/c and NOD/Lt mice to generate mAb specific for the FcγRI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected FcγRI encoded by the more common Fcgr1a and Fcgr1b alleles, and although they identified different epitopes, none inhibited the binding of IgG to FcγRI. When bound to FcγRI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of FcγRI. Resting and IFN-γ-induced macrophages expressed FcγRI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed FcγRI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of FcγRI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8α, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, FcγRI on APC may act as a link between innate and adaptive immunities by binding and mediating the uptake of T-independent immune complexes for presentation, thereby assisting in the development of T-dependent immune responses.

Foundational concepts and underlying theories for majors in “biochemistry and molecular biology”

Over the past two years, through an NSF RCN UBE grant, the ASBMB has held regional workshops for faculty members and science educators from around the country that focused on identifying: 1) core principles of biochemistry and molecular biology, 2) essential concepts and underlying theories from physics, chemistry, and mathematics, and 3) foundational skills that undergraduate majors in biochemistry and molecular biology must understand to complete their major coursework. Using information gained from these workshops, as well as from the ASBMB accreditation working group and the NSF Vision and Change report, the Core Concepts working group has developed a consensus list of learning outcomes and objectives based on five foundational concepts (evolution, matter and energy transformation, homeostasis, information flow, and macromolecular structure and function) that represent the expected conceptual knowledge base for undergraduate degrees in biochemistry and molecular biology. This consensus will aid biochemistry and molecular biology educators in the development of assessment tools for the new ASBMB recommended curriculum.

Biochemical evidence for a separate, MHC-linked locus encoding H-2.28 antigens.

In comparing the tryptic peptide maps of the H-2L and H-2D glycoprotein antigens isolated from NP-40 lysates of RADA1 (H-2a) leukemic cells, no more than 37% of the observed arginine-containing tryptic peptides are found to be homologous. Thus, the primary amino-acid sequences of these two antigens are probably less than 90% homologous. This constitutes the strongest evidence to date that the MHC-linkedH-2L region encodes H-2L antigens separately from theH-2D region, even though H-2L antigens bear D-end-associated antigenic determinants of the H-2.28 family. The anti-H-2.28 alloantiserum (k×r anti h2) used to precipitate H-2L antigens in this investigation was the NIH contract antiserum D28b. As the tryptic peptide maps also surprisingly revealed, D28b precipitates H-2D antigens as well and, thus, anti-H-2.4 immunoadsorbants were employed to isolate H-2L free of H-2D antigens. In light of the dual specificity of D28b, its reactivity with BALB/c-H-2dm2 mutant cells was re-examined. Even though mutant lymphocytes, which lack H-2L but not H-2D antigens, are not cytotoxically lysed by D28b (as are parental H-2d cells), D28b appears to precipitate H-2D antigens from NP-40 extracts of mutant splenocytes.

Using inquiry‐based exercises and interactive visuals to teach protein structure/function relationships

To help undergraduate biochemistry students gain deeper insights into the intricate relationships between protein structures and their biological functions, an instructional website (tutor.lscf.ucsb.edu/instdev/sears/ biochemistry) has been developed at the University of California, Santa Barbara with various hands-on, inquirybased learning exercises that are designed to challenge the critical thinking skills of students and, at the same time, evoke the scientific processes of data discovery, data analysis, inference making, and hypothesis testing. The website stages various activities so that students are exposed gradually to increasingly more complex structural and functional concepts over a 10-week period. For example, students begin their discovery of protein structure first by analyzing Chime-rendered images of standard and nonstandard amino acid structures, then simple peptides, and finally complex protein and enzyme structures. Likewise, students begin their discovery of protein functional data first by analyzing interactive Excel charts displaying data for simple monovalent ligand binding systems (e.g. buffers), then multivalent ligand binding systems with noninteracting or interacting binding sites (e.g. hemoglobin), and finally enzyme kinetic reactions in the absence or presence of inhibitors. To practice the process of making rational connections between structural and functional data, students regularly answer questions of a scientific nature on self-guided tutorials, self-grading practice quizzes, and graded examinations. For example, students might be asked to formulate or select a hypothesis about the properties of the microenvironment surrounding an ionizable group in a protein structure and then draw inferences about pK shifts that might be observed for that group under various experimental conditions; students might also be asked to predict changes in ligand binding data based on structural alterations or alterations in the microenvironment surrounding a ligand binding site. Almost all of the learning exercises at the instructional website have been created with standard, off-the-shelf programs or multimedia-based software tools, but a few customizations or software enhancements have been adopted to improve the interactive interface with the student. One enhancement, for example, greatly improves structure analysis using the popular Chime web browser plug-in; most web pages displaying Chime-rendered images include JavaScript-encoded “command entry” and “message recall” boxes (tutor.lscf.ucsb.edu/instdev/sears/biochemistry/ tw-enz/crbncanhydrase/carbonicanhydrasef.htm/), which enable students to employ a full range of RasMol commands for structure analysis including the default set of commands found in the Chime popup window. A second enhancement facilitates function analysis greatly with Excel (tutor.lscf.ucsb.edu/instdev/sears/biochemistry/sprdshts/hb-mb01-p.xls) or Microsoft Office Web Component (MSOWC) data charts (tutor.lscf.ucsb.edu/instdev/sears/ biochemistry/charts/phsat.hta); the interactivity of these data charts has been enhanced by adding adjustable buttons (or slider bars) that allow students to vary constants or data values systematically and interactively during analysis. A third enhancement involves the addition of some relatively simple Visual Basic (VB) code to active server page (ASP) practice quizzes that effectively makes them self-grading tests allowing students to gain instant feedback on their question responses (tutor.lscf. ucsb.edu/instdev/sears/biochemistry/practicequizzes/ prqz21/prqz21f.htm.) Because it is essential to evaluate this highly interactive learning environment to help determine whether the critical thinking skills of students are actually engaged and improved by the multimedia exercises, an ongoing longitudinal assessment has been initiated where student performance is evaluated at three stages, at the start of instruction, 10 weeks later when instruction ends, and four months after the end of instruction. Preliminary results indicate that although students initially have little skill in analyzing protein structure/function relationships, they gain the necessary computer and logic skills to connect important structure/function concepts by the end of instruction, and they also retain these skills (at somewhat diminished capacity) long after instruction is over. ‡ To whom correspondence should be addressed. Tel.: 805893-3499; Fax: 805-893-4724; E-mail: sears@lifesci.ucsb.edu.

DIFFERENTIAL EXPRESSION OF THE ASGM1 ANTIGEN ON ANTI-REOVIRUS AND ALLOREACTIVE CYTOTOXIC T LYMPHOCYTES (CTL)

Anti‐reovirus cytotoxic effectors were found to be: (i) H‐2 restricted; (ii) virus specific; (iii) non‐lytic (in 4 h) for natural killer (NK)‐sensitive YAC‐1 cells; and (iv) positive for the Thy‐1 and Lyt‐2 lymphocyte markers. Thus, anti‐reovirus cytotoxic effectors have the functional and phenotype characteristics of cytotoxic T lymphocyte (CTL). A significant fraction of anti‐viral CTL, as well as alloreactive CTL, were also found to be positive for the asialo GM1 (ASGM1) cell surface antigen, generally considered to be a NK cell marker. ASGM1 expression on these CTL, as determined by sensitivity to antibody plus complement (C), appeared to be highly variable and dependent on two factors—the nature of the antigenic stimulus (viral vs. alloantigen), and the mouse strain from which the CTL originated. Thus, ASGM1 antigen expression on CTL appears to be regulated and may be under the control of lymphokines, development differentiation signals and/or other strain‐dependent genetic factors.

Reversible ligand binding reactions: Why do biochemistry students have trouble connecting the dots?

Adaptive chemical behavior is essential for an organisms function and survival, and it is no surprise that biological systems are capable of responding both rapidly and selectively to chemical changes in the environment. To elucidate an organisms biochemistry, its chemical reactions need to be characterized in ways that reflect the normal physiology in vivo. This is a challenging experimental problem because biological systems are inherently complex with myriads of interlinked chemical networks orchestrating processes that are mostly irreversible in nature. One successful approach for simplifying the study of biochemical reactions is to analyze them under controlled reversible equilibrium conditions in vitro that approximate the range of physiological conditions found in vivo. Because this approach has helped elucidate some of the chemical mysteries of complex biological systems, many topics presented in modern biochemistry courses are essentially rooted in the chemistry of reversible equilibrium reactions. Since most undergraduate biochemistry courses typically require students to complete year‐long general and organic chemistry courses, biochemistry instructors may assume that entering students have sufficient understanding of basic reversible equilibrium chemistry to move forward into more advanced biochemical topics. However, this assumption is at odds with our experience in that many entering students seem confused by the conventions, language, symbolic formalism, and/or mathematical tools normally use to describe reversible equilibrium reactions. Part of the problem here may stem from how certain basic chemical concepts are taught (or are not taught) in their prerequisite chemistry courses. Here, we identify some conceptual barriers that many students seem to confront and we discuss instructional strategies designed to help students “connect the dots,” so to speak, and better understand how dynamic biological processes can be analyzed in terms of reversible equilibrium chemistry.

Mouse macrophage β subunit (CD11b) cDNA for the CR3 complement receptor/Mac-1 antigen

The cDNA for the common \Mac-1 subunit (CD11b) of the mouse LFA-1/Mac-1/p150,95 group of leukocyte cell adhesion receptors, formally designated integrin \2, has been cloned and sequenced. Clones were isolated from cDNA libraries made from J774 macrophage and WEHI-3B myelomonocytic tumor cells which express this subunit as a component of the macrophage activation antigen 1 (Mac-1), also known as complement receptor type 3 (CR3). This subunit is expressed as a single, abundant mRNA species approximately 2.7 kilobase (kb) in size. The 2422 base pair (bp) cDNA sequence obtained codes for a 771 amino acid protein organized with leader, extracellular, transmembrane, and cytoplamic domains of 23, 680, 23, and 46 amino acids, respectively, yielding an 82700 mature protein of 747 amino acids. The mouse \Mac-1 subunit is highly similar to its human counterpart with an overall sequence identity of 81% and identical positioning of 5 out of 6 potential N-linked glycosylation sites, as well as 56 Cys residues that are organized in repeating motifs characteristic of integrin \ subunits. The most highly conserved regions are the transmembrane and cytoplasmic domains where only 4 out of 69 amino acids differ, indicating that the functions associated with this domain in Mac-1-mediated processes, such as iC3b-triggered phagocytosis, have been evolutionarily conserved.

Unexpected complexity of the dm1 mutation revealed in the structure of three H-2D/L-related antigens

The H-2Ldm1 and H-2Ddm1 MHC antigens of the B10.D2 (H-2dm1) mutant mouse strain (formerly known as M504 or H-2da) have been compared to the H-2Ld and H-2Dd antigens of the B10.D2 (H-2d) mouse strain. Ldm1 and Ld are 45 000 Mr antigens and both are reactive with anti-H-2.“28” (k/r anti-h2) serum and unreactive with anti-H-2.4 (k/b anti-a) serum which detects private determinants of the Ddm1 and Dd antigens. However, the tryptic peptide compositions of these two antigens are different and, based on the number of major tryptic peptides which coelute during ion-exchange chromatography, the estimated peptide homology between Ldm1 and Ld is 80 percent. A newly defined antigen (Mr = 39 000), designated gp39dm1, was found in glycoprotein extracts of the dm1 strain but not of the d strain. This antigen coprecipitates with Ldm1 but does not coprecipitate with Ddm1 indicating that it lacks the H-2.4 determinant. In comparison with Ldm1, gp39dm1 appears to contain far fewer Arg and Lys residues and is most likely not a simple proteolytic fragment of Ldm1. Finally, peptide maps of the Ddm1 antigen show that the majority of its Arg peptides are identical to Dd Arg peptides, whereas at least five of its Lys peptides and three of its Arg peptides correspond not to Dd peptides but to Ld and Ldm1 peptides. These data raise the possibility that the Ddm1 antigen is a hybrid molecule and they have also revealed an unexpected level of complexity in the dm1 mutant phenotype.

Mechanism of Rapid Tumor Lysis by Human ADCC: Mediation by Monoclonal Antibodies and Fragmentation of Target Cell DNA

Immunologically programmed cell destruction is an important defense mechanism for limiting the spread of invasive intracellular parasites and malignant cells in the body (1,2). However, when such processes are misdirected, in autoimmune diseases for example, they can be very harmful to normal tissues and can interfere with vital tissue functions (2). Thus, it is of considerable value to understand the mechanisms of cytolytically induced cell destruction as mediated by the immune system. Several such mechanisms have evolved with the immune system of man, and each is characterized by distinct antigen recognition requirements and/or distinct lytic processes. Some cytolytic mechanisms require only the secreted products of cells and not the direct intervention of an effector cell for cytolysis to occur, as with complement plus antibody-mediated cytotoxicity (CAMC), for example. Other mechanisms, however, do require that intimate contact be made between an effector cell and its target cell. Various lymphocytes, such as cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and killer (K) cells all appear to initiate the lytic process only after contact is made with the target cell (1–4).

Biochemical evidence for structurally distinct H-2Dd antigens differing in serological properties

H-2Dd antigens, as defined by the private H-2.4 determinant, exist as two immunochemically distinct populations in H-2a and H-2dm2 splenocytes and in the transformed cell line, RADA1(H-2a). The two populations are distinguishable by the anti-H-2.28 serum, k/r anti-h2, which is directed, in part, against the H-2.28 family of public determinants encoded by the D end of the b haplotype. Sequential precipitates of lentil-lectin-purified glycoprotein extracts metabolically labeled with radioactive amino acids reveal that approximately one-quarter to one-third of the H-2Dd antigens, designated H-2Dd (b28+), react with this antiserum, whereas two-thirds to three-quarters, designated H-2Dd(b28−), do not. Paired-label tryptic peptide maps in this and a previous study indicate that H-2Dd(b28+) and H-2Dd(b28−) are closely related structurally and are more likely to represent modified forms of the same gene product rather than products of different genes, although the existence of closely related genetic loci is not rigorously excluded. Together, H-2Dd(b28+) and H-2Dd(b28−) have a radioactivity level seven times higher than H-2Ld, which also reacts with the anti-H-2.28 serum but which lacks the H-2.4 determinant. As yet unresolved, however, is the question of whether the observed quantitative differences between these three antigens reflect actual molar differences at the cellular level, or whether the variation is the result of metabolic or compositional factors. In any case, a complex serological and structural relationship is found to exist between antigens encoded by the D/L end of the MHC.