Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy.

We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.

Adjacent codon-anticodon interactions of both tRNAs present at the ribosomal A and P or P and E sites.

A labeled tRNA present at the A, P or E site can be partially chased from the ribosome, a cognate nonlabeled tRNA as chasing substrate being 3-12-times more efficient than non-cognate tRNA at a molar ratio tRNA: 70 S = 10:1. These findings indicate that a tRNA bound to a programmed ribosome undergoes codon-anticodon interaction at all three sites (A, P and E site). Furthermore, both labeled tRNA present on the ribosome can be chased more effectively with cognate than with non-cognate substrate at the same time. This finding provides strong evidence that both tRNAs present on the ribosome exhibit simultaneous codon-anticodon interaction. This is valid for both the pretranslocational state (Ac[3H]Lys-tRNALys in the A and [14C]tRNALys in the P site) as well as the posttranslocational state (Ac[3H]Lys-tRNALys in the P and [14C]tRNALys in the E site).

The function of the translating ribosome: allosteric three-site model of elongation.

During the last decade, a new model for the ribosomal elongation cycle has emerged. It is based on the finding that eubacterial ribosomes possess 3 tRNA binding sites. More recently, this has been confirmed for archaebacterial and eukaryotic ribosomes as well, and thus appears to be a universal feature of the protein synthetic machinery. Ribosomes from organisms of all 3 kingdoms harbor, in addition to the classical P and A sites, an E site (E for exit), into which deacylated tRNA is displaced during translocation, and from which it is expelled by the binding of an aminoacyl-tRNA to the A site at the beginning of the subsequent elongation round. The main features of the allosteric 3-site model of ribosomal elongation are the following: first, the third tRNA binding site is located 'upstream' adjacent to the P site with respect to the messenger, ie on the 5'-side of the P site. Second, during translocation, deacylated tRNA does not leave the ribosome from the P site, but co-translocates from the P site to the E site--when peptidyl-tRNA translocates from the A site to the P site. Third, deacylated tRNA is tightly bound to the E site in the post-translocational state, where it undergoes codon--anticodon interaction. Fourth, the elongating ribosome oscillates between 2 main conformations: (i), the pre-translocational conformer, where aminoacyl-tRNA (or peptidyl-tRNA) and peptidyl-tRNA (or deacylated tRNA) are firmly bound to the A and P sites, respectively; and (ii), the post-translocational conformer, where peptidyl-tRNA and deacylated tRNA are firmly bound to the P and E sites, respectively. The transition between the 2 states is regulated in an allosteric manner via negative cooperatively. It is modulated in a symmetrical fashion by the 2 elongation factors Tu and G. An elongating ribosome always maintains 2 high-affinity tRNA binding sites with 2 adjacent codon--anticodon interactions. The allosteric transition from the post- to the pre-translocational state is involved in the accuracy of aminoacyl-tRNA selection, and the maintenance of 2 codon--anticodon interactions helps to keep the messenger in frame during translation.

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.

Ephestia: the experimental design of Alfred Kühn'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 Kühn's 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.

[Carl Correns' experiments with Pisum, 1896-1899]. / Carl Correns' Experimente mit Pisum, 1896-1899.

The circumstances under which classical genetics became established at the turn of the nineteenth century have become an integral part of the standard narrative on the history of genetics. Yet, despite considerable scholarly efforts, it has remained a matter of debate how exactly the so-called 'rediscovery' of Mendel's laws came about around 1900. In this situation, unpublished research records can be invaluable tools to arrive at a more substantial and more satisfying picture of the order of historical events. This paper makes extended use of the research protocols covering Carl Correns' hybridisation experiments with Pisum sativum between 1896 and 1899. The resulting reconstruction sketches the portrait of a scientist following a particular research question--xenia--struggling with his experimental material, and slowly building up an epistemic regime in which questions and observations could acquire a relevance which did not strike Correns when he first took note of them. The microhistorical gaze through the magnifying glass of research notes reveals the kind of delays that appear to be constitutive for empirically-driven thinking in general. The research notes of Correns help not only to make this point, they also display some of the intricacies and material peculiarities which characterise the experimental process of hybridisation and the particular type of inferences it allows one to make.

Mendelian inheritance in Germany between 1900 and 1910. The case of Carl Correns (1864-1933).

Carl Correns (1864-1933) came to recognize Mendel's rules between 1894 and 1900 while trying to find out the mechanism of xenia, that is, the direct influence of the fertilizing pollen on the mother plant in maize and peas among other species. In this paper, I am concerned with the ten years of Correns' work after the annus mirabilis of 1900 until 1910, when the main outlines of the new science of genetics had been established. It is generally assumed that after 1900 Correns quickly began probing the limits of Mendelian inheritance, both as far as the explanatory force of formal transmission genetics and the generality of Mendel's laws are concerned. A careful examination of his papers however shows that he was much more interested in the scope of Mendelian inheritance than in its limits. Even his work with variegated Mirabilis plants, which historiographical folklore still presents as a result of Correns' growing interest in cytoplasmic inheritance, can be shown to have been conducted to corroborate just the opposite, namely, the validity of the nuclear paradigm. The paper will show that Correns' research results in those years (among them the Mendelian inheritance of sex in higher plants) were the outcome of a complex experimental program which involved breeding experiments with dozens of different species.

[Buffon: time, change and history]. / Buffon: Zeit, Veränderung und Geschichte.

There is a longstanding and ongoing controversy about whether Buffon is to be regarded as a forerunner of evolutionism in the eighteenth century, or even as one of the founders of transformistic biology. There are good reasons to deny this claim. There are good reasons even to deny that the question which is going to be answered negatively is of particular importance. The present paper addresses the issue from a different angle. It analyzes the concept of time operative in the natural history writings of Buffon, and it delineates the articulation of the concepts of time, change, and history with its organizing impact on Buffon's discourse on earth and organisms. It is argued that although with his species concept Buffon tries to introduce the classical notion of a physical system into biology, in order to do so, he has to subvert it by an element of time. This guides him in considering various aspects of organic change, but by itself does not lead to a general perspective of transformation. On the other hand, in his Epoques de la nature, Buffon introduces a general law of geological change, thus arriving at something which could be called a physically intelligible history. The conquest of natural history by physics, in one and the same movement, leads to a subversion of physical geology by history, and prevents biology from becoming evolutionistic in the sense in which the nineteenth century understands this term.

Beyond nature and culture: a note on medicine in the age of molecular biology.

This paper is divided into two parts. In the first, I examine the relations among molecular biology, gene technology, and medicine, as well as some aspects of the consequences of these relations with respect to the human genome project. I argue that the prevailing momentum of early molecular biology resided in creating the technical means for an extracellular representation of intracellular configurations. As such, its medical impact was rather limited. With the advent of recombinant DNA technologies, a radical change of perspective ensued. The momentum of gene technology is based on the prospects of an intracellular representation of extracellular projects--the "rewriting" of life. Its medical impact is potentially unlimited. In the second part, I question the very opposition between nature and culture that implicitly underlies the notion of medicine as a "cultural system." I argue that both on a macroscopic level (global ecological changes) and on a microscopic level (genetic engineering), the "natural" and the "social" are no longer to be seen as ontologically different. In its uncanny oscillation between retrospection and foresight, between description and proclamation, and between assertion and hesitatiion, this essay translates an uneasiness that I have not been able to overcome while writing it. The essay conveys the tangled views of a hybrid author who himself cannot but oscillate between the perspectives of an actor in the field of molecular biology, a participant in the field of science studies, and a citizen.

Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol.

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).

Allosteric interactions between the ribosomal transfer RNA-binding sites A and E.

We have previously proposed a three-site model for the elongation cycle. The model is characterized by the presence of two tRNAs on the ribosome before and after translocation. We have already shown a first consequence of the model, namely that the translocation reaction is not coupled with a release of deacylated tRNA. Here we demonstrate the following conclusions. Occupation of the A site triggers the tRNA release from the E site, i.e. the A site occupation induces a drastic decrease in the affinity of the E site for deacylated tRNA. In the concentration range of deacylated tRNA in which a ribosome binds a second tRNA in addition to that one already present at the P site the deacylated tRNA does not compete for one and the same binding site with an A site ligand (AcPhe-tRNA) at 37 degrees C. It follows that the second deacylated tRNA binds to a site, the E site, which is physically distinct from the A site. When the ribosome binds a deacylated tRNA at the E site (in addition to a tRNA at the P site), the A site cannot be occupied by AcPhe-tRNA at 0 degree C and only poorly by the ternary complex elongation factor Tu . Phe-tRNA . guanyl-5'-yl imidodiphosphate. At 37 degrees C a significant A site binding is observed, with a corresponding tRNA release from the E site. In contrast, if the E site is free and only the P site occupied, the A site can bind significant amounts of charged tRNA already at 0 degree C. It follows that an occupied E site induces a low-affinity state of the A site. Thus, the ribosome always contains two high-affinity binding sites, which are A and P sites before and P and E sites after translocation. A and E sites are allosterically linked in a bidirectional manner.

Testing an alternative model for the ribosomal peptide elongation cycle.

A kinetic analysis of poly(U)-dependent poly(Phe) synthesis with [14C]tRNAPhe and [3H]phenylalanine demonstrated that, in the course of efficient poly(Phe) synthesis, two tRNAs are present per 70S ribosome at all times, although at least 70% of the poly(Phe)-tRNAPhe is found at the peptidyl-tRNA (P) site. Together with our recent observation of a third tRNA-binding site on Escherichia coli ribosomes, these findings suggest a model for the peptide elongation cycle in which two tRNA molecules are present on the ribosome at both the pre- and the post-translocational state. This model predicts that deacylated tRNA is not released from the P site but translocated to the exit (E) site before release occurs. A series of translocation experiments with deacylated [14C]tRNAPhe at the P site and oligo [( 3H]Phe)-tRNA at the aminoacyl-tRNA (A) site proved that efficient elongation factor G-dependent translocation is not accompanied by a corresponding [14C]tRNAPhe release. However, significant [14C]tRNAPhe release was observed after translocation when an aminoacyl-tRNA was bound to the A site. Thus, deacylated tRNA is not released from the P site but is translocated to the E site, which therefore must be located "upstream" adjacent to the P site. Furthermore, the trigger for the release of deacylated tRNA from the E site is the binding of aminoacyl-tRNA to the A site.