Co-expression of an epitope on human free kappa-light chains and on a cytoplasmic component in activated T cells

K-1-21 is a murine monoclonal antibody that reacts with human kappa-light chains in free form but not when they are associated with immunoglobulin heavy chains. K-1-21 was unexpectedly shown to bind to a determinant, STA (Sezary T cell antigen), detected by immunofluorescence in the cytoplasm but not on the surface of Sezary T cells isolated from peripheral blood (4/4 cases) and in Sezary T cells from lymph node and bone marrow (one patient). STA was detected in F2/F7, CCRF-CEM, Molt-4, and CCRF-HSB (four human T ALL cell lines), in JURKAT (a human T cell leukemia line), and in MLA144 (a Gibbon T cell lymphoma line). It also occurred in Leu-3a+ antigen-specific T cell clones (6/6 tested). Moreover, although STA was absent from freshly isolated normal T cells, its expression could be evoked in E+ cells from peripheral blood by in vitro culture with phytohemagglutinin. Thus, STA appears to be a cytoplasmic marker for activated T cells. Cytoplasmic inhibition immunofluorescence studies indicated that K-1-21 binding to STA in Sezary cells or T cell lines was inhibited by preincubation of the K-1-21 antibody with purified kappa-Bence Jones protein. STA from radiolabeled MLA144 cell lysates was immunoprecipitated by K-1-21 and was identified on polyacrylamide gel electrophoresis under reducing conditions as a protein of m.w. 57,000. Additional experiments are underway to define the molecular basis of the interesting cross-reactivity between a determinant in T cells and the K-1-21 reactive epitope on free kappa-light chains.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

The molecular basis of the requirement for antigen processing of pigeon cytochrome c prior to T cell activation

Antigen-induced activation of T lymphocytes that co-recognize Ia molecules has been shown to require an antigen-processing step by the presenting cell before T cell stimulation can occur. In this report, we demonstrate that antigen presentation of pigeon cytochrome c to an E kappa beta:E kappa alpha-restricted T cell hybridoma, 2C2, is inhibited by pretreatment of the antigen-presenting cells (APC) either with chloroquine or with fixation by paraformaldehyde. The chloroquine effect was partially reversible after 22 hr; the paraformaldehyde effect was not. In contrast, these treatments had little or no effect on the presentation of the carboxy-terminal cyanogen bromide cleavage fragment of pigeon cytochrome c, residues 81 to 104. There was at least a 50-fold greater potency of the fragment, as compared to that of the intact molecule, when paraformaldehyde-fixed APC were used. In addition, the fixed cells did not present synthetic fragments of the cytochrome c that were nonstimulatory when presented by unfixed cells. This observation showed that the loss of potency, demonstrated previously for analogs of pigeon cytochrome c with single amino acid substitutions at positions such as 99, was not a consequence of an alteration in the rate of antigen-processing. This result is consistent with our earlier hypothesis that these residues are contact amino acids with the antigen-specific T cell receptor or the Ia molecule. The major goal of these experiments was to define the molecular transition that occurred as a result of antigen processing. To achieve this end, we tested a variety of pigeon cytochrome c molecules and fragments for their ability to be presented by paraformaldehyde-fixed APC. Apocytochrome c, the denatured form of the molecule with the heme removed, could not be presented by the fixed cells, nor could the fragment 60-104, derived by acid cleavage of the tryptophan at position 59. Both molecules stimulated an IL 2 response from the T cell hybridoma when unfixed APC were utilized, demonstrating that the conditions used to prepare these two molecules did not destroy their antigenic determinant. In contrast, carboxy-terminal fragments, both native and synthetic, ranging in size from 16 to 39 amino acids, were capable of stimulating in the presence of paraformaldehyde-fixed APC. In particular, the partial-digest cyanogen bromide fragment, residues 66 to 104, was only twofold less potent than the pigeon fragment 81-104.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Radiation chemistry of a multicomponent aqueous system relevant to chemistry of cometary nuclei

Summary We have examined a water-dominated multicomponent system after irradiation in the multimegarad dose range with gamma rays from a⁶⁰Co source at both 77 and 310 K. The constituents were simple organic compounds in the proportions in which they appear in a dense interstellar cloud: HCN/CH₃OH/CH₃CN/C₂H₅CN/HCOOH=10.60.20.10.05. The total amounts were adjusted to correspond to a carbon to nitrogen ratio of 1.8 and a water content of about 50% in a cometary nucleus where the dust to volatiles ratio is 1; the total amount of CN-bearing compounds was taken to correspond to 0.4% of the cometary mass. In experiments at 310 K about 40 radiolytic products are identified, among them aldehydes and amino and carboxylic acids. Abundant polymeric material (M_w up to 80,000 daltons) is formed. The basic aspects of radiolysis of the liquid system are present also at 77 K, although at radiation-chemical yields that are lower by one to two orders of magnitude. We have considered the relevance of the present findings to the chemistry of a liquid-water core and the icy layers of a cometary nucleus.     

Radiation chemistry of an aqueous solution of glycine: compounds of interest to chemical evolution studies

Summary We have examined the radiolysis of an O₂-free aqueous solution of glycine at absorbed doses of⁶⁰Co gamma-radiation of up to 20 Mrad. At least 20 compounds are formed during radiolysis, among them several amino acids, an oligoamine, and the nitrogen-free polymers (M_w28,000 daltons). When dicyandiamide is present in the solution of glycine, various nitrogen-containing products, including some polymers (M_w12,000 daltons), are synthesized along with radiolytic products of glycine; polyglycines are not formed. We have determined the radiation-chemical yields of radiolytic-product formation and of decomposition of glycine, and have considered possible free-radical reactions leading to the radiation-induced changes observed.     

Changes in LH and prolactin levels in diabetic male rats and the role of the opiate system in the control of their secretion

Diabetic male rat has low serum levels of luteinizing hormone (LH) and testosterone (T), which are accompanied by atrophy of the testes and accessory glands. The present study investigated changes in the serum levels of LH, prolactin (PRL) and glucose, following diabetes induction by streptozotocin. In addition, involvement of the opiate system in the control of LH and PRL secretion was evaluated. There was no difference in PRL levels between diabetic and control animals, except at 8 hours after streptozotocin injection. In contrast, the diabetic animals had consistently lower levels of LH, starting on the second day of diabetes. Blockade of the opiate system by naltrexone caused a sharp increase of LH levels in normoglycemic rats, while only a gradual decrease was observed in hyperglycemic animals. PRL secretion was inhibited by naltrexone, both in diabetic and control groups. It is concluded that, unlike normoglycemic rats, inhibition of LH secretion in diabetes is not under the control of the opiate system, probably as a result of T deficiency. In contrast, PRL secretion in diabetic rats, as in the control group, is under the influence of endogenous opiates.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.