Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induce IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine σ-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.     

Isolation and RNA-binding analysis of NAD+-isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe

Krebs cycle NAD⁺-isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiaeCOX2 leaders. In contrast, Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA. Received: 19 January 2000 / 24 March 2000     

Molecular identification of T cell-specific antigens on human T lymphocytes and thymocytes

We have identified membrane glycoproteins which carry T cell-specific antigens on human T lymphocytes and thymocytes. Purified cells were surface-labeled with NaB³H₄ after treatment with neuraminidase and galactose oxidase. Immunoprecipitations were performed with rabbit anti-human T cell-specific antibodies using co precipitation with protein A-containing staphylococci strain Cowan I. The labeled membrane glycoproteins and the precipitates were subjected to polyacrylamide slab gel electrophoresis and visualized by fluorography. The antibodies specifically precipitated 4 proteins called GP200, GP180, GP165 and GP160 (mol. wts. = 200000, 180000, 165000 and 160000) from surface-labeled T lymphocytes and low-density (medullary) thymocytes. The GP200 and GP180 were not labeled on high-density (cortical) thymocytes. A protein with a mol. wt. of 45000 was precipitated from thymocytes. Another glycoprotein on T lymphocytes and thymocytes with a mol. wt. similar to that of mouse and rat Thy-1 or Θ antigen (mol. wt. 25000) reacted with the antibodies.     

Structural Reasons for Restricted Backbone Motion in Poly(n‐alkyl methacrylates): Degree of Polymerization,Tacticity and Side‐Chain Length

Summary: The complex dynamics of poly(n‐alkyl methacrylates) is studied by advanced ¹³C NMR spectroscopy as well as mechanical and dielectric relaxation. Extended backbone conformations are identified as the molecular units involved in structural relaxation. From the variation in the degree of polymerization and a comparison with the presence of stereoregular sequences in the sample, the length of the extended units is determined to involve about five, at most ten monomeric units. Syndiotactic and isotactic sequences behave similarly. These findings are indicative of locally structured polymer melts.

     

Crystallization and Melting Behavior of Poly(3‐hydroxybutyrate)‐Based Blends

Summary: Blends of high molecular weight poly(R‐3‐hydroxybutyrate) (PHB) ( = 352 000 g · mol^?1), comprising of either low molecular weight poly(R‐3‐hydroxybutyrate) (D‐PHB) ( = 3 900 g · mol^?1) or poly[(R‐3‐hydroxybutyrate)‐co‐(R‐3‐hydroxyvalerate)] (PHBV) ( = 238 000 g · mol^?1) with 12 mol‐% hydroxyvalerate (HV) content as a second constituent, were investigated along with the thermal properties and morphologies. After isothermal crystallization, a lowering of the melting temperature of PHB can be observed with increasing content of the second component in the blends. This behavior points towards miscibility of the constituents both in the liquid and the solid state. Crystallization kinetics was studied under isothermal and non‐isothermal conditions. The overall kinetics of isothermal crystallization was analyzed in terms of the Avrami equation. Only one crystallization peak is observed in all cases for the PHB/D‐PHB and PHB/PHBV blends under the conditions studied. This demonstrates co‐crystallization of the constituents. The addition of D‐PHB or PHBV to PHB reduces the rate of crystallization of the blends compared to that of neat PHB. The corresponding activation energies of crystallization also decrease with an increasing concentration of the second constituent. Non‐isothermal crystallization, carried out with different cooling rates held constant, is discussed in terms of a quasi‐isothermal approach. The corresponding rate constants as functions of reciprocal undercooling show Arrhenius‐like behavior in a certain range of temperatures. At sufficiently high undercooling, the rate constants of crystallization for the isothermal process exceed those reflecting non‐isothermal conditions, whereas in the limit of low undercoolings, the rate constants become similar. Ring‐banded morphologies are observed when PHB is in excess. When the respective second component is the major component, fibrous textures of the spherulites develop.

Polarized micrograph of PHB/PHBV 90/10.     

Extracellular detection of K+ release during migration of transformed Madin-Darby canine kidney cells

 Madin Darby canine kidney cells transformed by alkaline stress (MDCK-F cells) constitutively migrate at a rate of about 1 μm·min^–1. Migration depends on the intermittent activity of a Ca²⁺-stimulated, 53-pS K⁺ channel (K_Ca channel) that is inhibitable by charybdotoxin. In the present study we examined whether this intermittent K_Ca channel activity results in a significant K⁺ loss across the plasma membrane. K⁺ efflux from MDCK-F cells should result in a transient increase of extracellular K⁺ ([K⁺]_e) in the close vicinity of a migrating cell. However, due to the rapid diffusion of K⁺ ions into the virtually infinite extracellular space, such a transient increase in [K⁺]_e was too small to be detected by conventional K⁺-selective electrodes. Therefore, we developed a ”shielded ion-sensitive microelectrode” (SIM) that limited diffusion to a small compartment, formed by a shielding pipette which surrounded the tip of the K⁺-sensitive microelectrode. The SIM improved the signal to noise ratio by a factor of at least three, thus transient increases of [K⁺]_e in the vicinity of MDCK-F cells became detectable. They occurred at a rate of 1.3 min^–1. The cell releases 40 fmol K⁺ during each burst of intermittent K_Ca channel activity, which corresponds to about 15% of the total cellular K⁺ content. Since transmembrane K⁺ loss must be accompanied by anion loss and therefore leads to a decrease of cell volume, these findings support the hypothesis that intermittent volume changes are a prerequisite for the migration of MDCK-F cells. Received: 15 April 1996 / Received after revision: 18 June 1996 / Accepted: 23 July 1996     

Characterization of FMR1 proteins isolated from different tissues

FMR1 protein expression was studied in different tissues. Inhuman, monkey and murine tissues, high molecular mass FMR1 proteins(67–80 kDa) are found, as shown in lymphobiastoid celiiines. The identity of these proteins was confirmed by theirabsence in tissues from patients with the fragile X syndromeand a FMR1 knock-out mouse. An IIe367Asn substitution in theFMR1 protein did not aiter the transiation, processing and localizationof FMR1 proteins in lymphoblastoid cells from a patient carryingthis mutation. All the high molecular mass FMR1 proteins isolatedfrom normal lymphoblastoid cells and cells from the patientwith the IIe367Asn substitution were able to bind RNA. However,the FMR1 proteins of the patient had reduced affinity for RNAbinding at high salt concentrations. In some human, monkey andmurine tissues low molecular mass FMR1 proteins (39–41kDa) were found, which had the same N terminus as the 67–90kDa isoforms, but differ in their C terminus and are thereforemost likely the result of carboxy-terminal proteolytic cleavage.These low molecular mass FMR1 proteins did not bind RNA, incontrast with the high molecular mass FMR1 proteins. The significanceof these low molecular mass proteins remains to be studied.     

Site-directed ELISA with synthetic peptides representing the HIV transmembrane glycoprotein

Two partially overlapping 19 and 22 amino acids long peptides representing a highly immunogenic site of the transmembranous glycoprotein (gp41) of human immunodeficiency virus (HIV) were used as antigen in ELISA tests. The results of antibody determination with this assay were compared with those of three or more conventional ELISAs and Western blot (WB) tests and radioimmunoprecipitation assay. Twenty-six sera from patients with AIDS or LAS and from asymptomatic carriers of HIV infection all showed a pronounced reaction in the peptide ELISA as well as positive results with other tests. In contrast, 27 sera from laboratory workers and blood donors were negative by all tests. A group of 39 blood donor sera, which had shown false positive or ambiguous results in the ELISAs and sometimes in WB tests employed for confirmation, also were negative in all cases with the peptide ELISA. Consecutive samples collected from individuals with primary HIV infection were also analyzed. In 6 out of 9 cases, the peptide ELISA revealed an antibody response within one month after onset of clinical symptoms and sensitivity for antibody detection equaled that of other ELISA tests. Eight sera from five West African persons infected with HIV-related viruses did not react in the peptide ELISA, reflecting differences in properties of the envelope components. The peptide ELISA used in this study appears to represent a simple technique employing chemically synthesized antigen for accurate and sensitive estimation of antibodies to the HIV group of nontransforming human retroviruses.     

Apparent superiority of H2-receptor stimulation and simultaneousβ-blockade over conventional treatment withβ-sympathomimetic drugs in post-acute myocardial infarction: Cardiac effects of impromidine — a new specific H2-receptor agonist — in the surviving catecholamine-insensitive myocardium

Left ventricular infarction (AMI) was produced in experimental animals and the contractile response to -adrenergic and H₂-histaminergic stimulation by isoproterenol and impromidine tested in the isolated perfused heart preparation. Adenylate cyclase activity as well as binding characteristics of [³H]-dihydroalprenolol ([³H]-DHA), [³H]-methyl-tiotidine ([³H]-TIOT) and [³H]-quinuclidinyl benzilate ([³H]-QNB) to cardiac ₁-, H₂- and cholinergic muscarinic receptors were determined in sarcolemmal membrane preparations of the right ventricle of the same hearts. In addition, an attempt was made to elucidate the therapeutic value of post-AMI treatment with impromidine in the presence and absence of-sympathomimetic, in contrast to administration of prenalterol and the conventional therapy with -sympathomimetic drugs, e.g. dobutamine. Three days post-AMI the dose-response curve for isoproterenol of right ventriculardP/dt _max was significantly depressed, while the inotropic effect of impromidine was not impaired. Stimulation of adenylate cyclase activity by isoproterenol was reduced by 80% whereas impromidine and NaF stimulation rates were unaltered. Receptor-binding studies indicated a 90% loss and 10-times lowered affinity (K _D) of the remaining -receptors while specific [³H]-TIOT- and [³H]-QNB-binding was unchanged.Administration of dobutamine increased mortality rates and extension of infarct size, led to a further decrease in contractile response to isoproterenol, induced complete insensitivity of adenylate cyclase to isoproterenol stimulation and caused pronounced additional reduction of number and affinity of [³H]-DHA-binding sites. In contrast, all above alterations were prevented by treatment with either prenalterol or combined administration of impromidine plus metoprolol. It is concluded, that these alterations in the non-ischemic, uninvolved myocardium post-AMI are the result of catecholamine-induced specific damage of sarcolemmal -receptors. Furthermore, treatment with H₂-agonists in combination with -blocking agents may have beneficial effects, whereas conventional therapy with -sympathomimetic drugs tends to worsen the already depressed function of the -adrenergic stimulation mechanism.Supported by grants Ba 666/1 and Ba 666/2-2 from the Deutsche Forschungsgemeinschaft (DFG).Data presented in this paper are part of a doctoral thesis by Dr S.B. Felix.