Hans-Jörg Rheinberger

Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE‐mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site‐directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple‐point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen‐allergic patients. In vivo (skin prick) tests showed that the potency of the multiple‐point mutant to induce typical urticarial type I reactions in pollen‐allergic patients was significantly lower than for wild‐type Bet v 1. Proliferation assays of allergen‐specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six‐point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate‐type allergies.—Ferreira, F., Ebner, C., Kramer, B., Casari, G., Briza, P., Kungl, A. J., Grimm, R., Jahn‐Schmid, B., Breiteneder, H., Kraft, D., Breitenbach, M., Rheinberger, H.‐J., Scheiner, O. Modulation of IgE reactivity of allergens by site‐directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J. 12, 231–242 (1998)

Immunological and Biological Properties of Bet v 4, a Novel Birch Pollen Allergen with Two EF-hand Calcium-binding Domains

We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.

Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding

Publisher Summary This chapter discusses a preparation procedure for tightly coupled 70S ribosomes of Escherichia coli, as well as for 30S and 50S subunits derived from tight couples. Highly active ribosomes are a prerequisite to characterize quantitatively the interactions between transfer RNA, messenger RNA, and ribosomal particles. Preparation procedures as well as the binding capacities of ribosomal particles for all three kinds of tRNA involved in the elongation cycle-peptidyl-tRNA, aminoacyl-tRNA, and deacylated tRNA have remained a matter of controversy until recently. The preparation procedure has been developed by modifying a protocol and it reproducibly yields a high level of active ribosomes. The chapter also describes the in vitro system which allows the site-specific ribosomal tRNA-binding activity to be tested. Further difficulties in evaluating ribosomal activity are based on the fact that: (1) there may be functional heterogeneity within a ribosomal population carrying a tRNA in a distinct site. Such heterogeneity would not be reflected in the mere binding numbers, and (2) tRNA-binding capacity and overall elongation activity need not necessarily coincide.

An epistemology of the concrete : twentieth-century histories of life

An Epistemology of the Concrete brings together case studies and theoretical reflections on the history and epistemology of the life sciences by Hans-Jorg Rheinberger, one of the world’s foremost philosophers of science. In these essays, he examines the history of experiments, concepts, model organisms, instruments, and the gamut of epistemological, institutional, political, and social factors that determine the actual course of the development of knowledge. Building on ideas from his influential book Toward a History of Epistemic Things , Rheinberger first considers ways of historicizing scientific knowledge, and then explores different configurations of genetic experimentation in the first half of the twentieth century and the interaction between apparatuses, experiments, and concept formation in molecular biology in the second half of the twentieth century. He delves into fundamental epistemological issues bearing on the relationship between instruments and objects of knowledge, laboratory preparations as a special class of epistemic objects, and the note-taking and write-up techniques used in research labs. He takes up topics ranging from the French “historical epistemologists” Gaston Bachelard and Georges Canguilhem to the liquid scintillation counter, a radioactivity measuring device that became a crucial tool for molecular biology and biomedicine in the 1960s and 1970s. Throughout An Epistemology of the Concrete , Rheinberger shows how assemblages—historical conjunctures—set the conditions for the emergence of epistemic novelty, and he conveys the fascination of scientific things: those organisms, spaces, apparatuses, and techniques that are transformed by research and that transform research in turn.

A cultural history of heredity

It was only around 1800 that heredity began to enter debates among physicians, breeders, and naturalists. Soon thereafter it evolved into one of the most fundamental concepts of biology. Here Staffan Muller-Wille and Hans-Jorg Rheinberger offer a succinct cultural history of the scientific concept of heredity. They outline the dramatic changes the idea has undergone since the early modern period and describe the political and technological developments that brought about these changes. Muller-Wille and Rheinberger begin with an account of premodern theories of generation, showing that these were concerned with the procreation of individuals rather than with hereditary transmission. The authors reveal that when hereditarian thinking first emerged, it did so in a variety of cultural domains, such as politics and law, medicine, natural history, breeding, and anthropology. Muller-Wille and Rheinberger then track theories of heredity from the late nineteenth century - when leading biologists considered it in light of growing societal concerns with race and eugenics - through the rise of classical and molecular genetics in the twentieth century to today, as researchers apply sophisticated information technologies to understand heredity. What readers come to see from this exquisite history is why it took such a long time for heredity to become a prominent concept in the life sciences and why it gained such overwhelming importance in those sciences and the broader culture over the last two centuries.

Crystallization of Escherichia coli ribosomes

The knowledge of the structure of the ribosome is an essential requirement to reveal its role at the molecular level in the process of protein biosynthesis. This information is being obtained by a battery of chemical, physical, immunological and genetic methods (for reviews see [1]). Important methods for the study of the three-dimensional structure of the ribosomes are X-ray crystallography and image reconstruction. Two-dimensional ordered sheets of ribosomes from a few eukaryotic species are found under special conditions in vivo [2-4] or in cell homogenates [5]. Small two-dimensional arrays of E. coli ribosomal subunits were obtained in vitro [6,7]. The first three-dimensional crystals of ribosomal particles were obtained in vitro from the large subunit of the Bacillus stearothermophilus ribosome [8,9]. These have been characterized by three-dimensional image reconstruction studies [10]. Here we report the in vitro crystallization of E. coli ribosomes. We have obtained ordered threedimensional microcrystals. After washing and redissolving, the crystalline ribosomes sediment with a coefficient of 70 S in a sucrose gradient and are biologically active in the poly(U) in vitro system. Electron micrographs of thin sections of the crystals show regular arrangement of the ribosomes, and their optical diffraction patterns extend out to about 6.5 nm. Thus three-dimensional image reconstruction studies can be carried out. 2. MATERIALS AND METHODS

From molecular genetics to genomics : the mapping cultures of twentieth century genetics

Section 1. Molecularizing Maps: 1. Angela Creager - Mapping Genes in Microorganisms 2. Frederic L. Holmes - Seymour Benzer and the Convergence of Molecular Biology with Classical Genetics 3. Marcel Weber - Walking on the Chromosome: Drosophila and the Molecularization of Development 4. Scott Gilbert -. Gene Expression Maps: The Cartography of Transcription 5. Soraya de Chadarevian - Mapping the Worms Genome: Tools, Networks, Patronage Section 2. The Moral and the Political Economy of Human Genome Sequencing: 6. Stephen Hilgartner - Making Maps and Making Social Order: Governing American Genome Centers, 1988-93 7. Alain Kaufmann - Mapping the Human Genome at Genethon Laboratory: The French Muscular Dystrophy Association and the Politics of the Gene 8. Adam Bostanci - Sequencing Human Genomes 9. Gisli Palsson -. Decoding Relations and Disease: The Icelandic Biogenetic Project

The ribosomal E site at low Mg2+ : coordinate inactivation of ribosomal functions at MG2+ concentrations below 10 mM and its prevention by polyamines

Under standard conditions (Mg2+/150 mM NH4+) ribosomes can quantitatively participate in tRNA binding at Mg2+ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P, E or A sites decrease in a parallel manner when the Mg2+ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg2+, and is completely blocked at 6-8 mM. The severe inactivation seen at 6 mM Mg2+ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg2+ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus, J. Biol. Chem. 261, 9133 (1986] are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites). The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg2+/NH4+/polyamine-system seems to be the best compromise at present between these two requirements.

A reply to David Bloor: 'Toward a sociology of epistemic things'

First of all, I would like to thank David Bloor for his very thoughtful reoections on the notion of “epistemic things.” He has surely pointed toward certain difaculties and even impasses that I have tried to do my best to circumvent in my book, but for which I was certainly not able to provide an ultimate solution. Let me then respond with a few remarks 1) on what it means to be an epistemic object; 2) on the problem of reference; 3) on the social constitution of the objects of science; and 4) on technoscience and the problem of demarcation. David contends that Toward a History of Epistemic Things faces, in his words, “a general problem about the nature of scientiac discourse, namely the problem of reference.” Reference, according to him, “is, or involves, aboutness.” His main critique addresses what he, in contrast, calls my “non-referentiality thesis.” Before I tackle this point, I think I should say a few words on the notion of “epistemic object.” The general thrust of my whole argument is about the power of material objects—in contrast to ideas or concepts—as driving forces in the process of knowledge acquisition. Consequently, I am somewhat surprised to and my work categorized, in David’s critique, under the label of “linguistic idealism.” My goal was to provide an objectcentered, materially founded account of knowledge production. According to my position, scientiac or epistemic objects are clearly material things. They function as scientiac or epistemic objects by virtue of their opacity, their surplus, their material transcendence, if you like, which is what arouses interest in them and keeps them alive as targets of research. The fact that referentiality is not what characterizes their essence does not, by any means, as I see it, catapult them into the realm of the ideal. They

Ephestia: The Experimental Design of Alfred Kuhn's Physiological Developmental Genetics *

Much of the early history of developmental and physiological genetics in Germany remains to be written. Together with Carl Correns and Richard Goldschmidt, Alfred Kühn occupies a special place in this history. Trained as a zoologist in Freiburg im Breisgau, he set out to integrate physiology, development and genetics in a particular experimental system based on the flour moth Ephestia kühniella Zeller. This paper is meant to reconstruct the crucial steps in the experimental pathway that led Kühn and his collaborators at the University of Göttingen, and later at the Kaiser Wilhelm Institutes of Biology and Biochemistry in Berlin, to formulate, in their specific way, what later became known as the “one gene – one enzyme hypothesis.” Special attention will be given to the interaction of the different parts of Kühns Ephestia-based project, which were rooted in different research traditions. The paper retraces how, roughly between 1925 and 1945, these elements came to form a mixed experimental set-up composed of genetic, embryological, physiological and, finally, biochemical constituents. Accordingly, emphasis is laid on the development of the terminology in which the results were cast, and how it reflected the hybrid state of an experimental system successively acquiring new epistemic layers.