Characterization of specific HLA-DQ alpha allospecificities by genomic, biochemical, and serologic analysis

Bgl II restriction endonuclease digestion of genomic DNA from lymphoblastoid cell lines homozygous for HLA DR and DQ serological specificities, followed by hybridization with a DQ alpha cDNA probe, identified a genomic polymorphism characterized by two reciprocal patterns, one associated with DR 3, 5 and 8 and the other with DR 1, 2, 4, 7, and 9. The former pattern corresponded precisely to the reactivity of monoclonal antibody SFR20-DQ alpha 5, shown by Western blotting to react with isolated alpha-chains, but not with beta-chains. Additional variants of the DQ alpha genes were identified by using a locus-specific oligonucleotide probe for the DQ alpha gene, indicating differences among the DQ alpha 5-negative set of alleles. This analysis defines a set of DQ alpha allelic markers that are distinct from the well-established DQ serologic specificities DQw1, 2, 3 or "blank." Although most DQ alpha 5+ cells carry the DRw52 specificity associated with the DR beta 2 gene, analysis of DQ alpha polymorphisms on DR5, DQw1; DR8, DQw1; and DRw13, DQw1 cells verified that this DQ alpha family of alleles was not invariably linked to the DR beta 2 locus.     

In vivo priming of helper and suppressor T cells by alloantigens. Frequency analysis with the use of an in vitro limiting dilution assay

Earlier studies have demonstrated that T cells activated in mixed lymphocyte reactions can exert positive as well as negative allogeneic effects on B cells expressing the appropriate alloantigens on their surface. We investigated the effect of in vivo priming of T cells with alloantigens on their capacity to help or suppress allogeneic B cell cultures against sheep erythrocytes. We used immunization protocols that have been shown to be optimal for induction of alloantigen-specific delayed-type hypersensitivity (DTH) and alloantigen-specific suppressor T (Ts) cells for DTH. The results show that in vivo stimulation with alloantigens, depending on the immunization route and the lymphoid organ studied, can be as effective as in vitro stimulation in increasing the frequency of alloantigen-specific helper T (Th) cells and Ts cells. Subcutaneous immunization induced a 10-fold frequency raise of Th cells as well as of Ts cells in the lymph nodes. In the spleen the Th cell population was hardly affected by s.c. immunization, whereas the Ts cell population increased by at least a factor 20. Intravenous immunization, on the other hand, selectively expanded the Th cell population in the spleen, whereas the splenic Ts cell population and the Th and Ts cells in the lymph nodes were not affected. Comparison of these results with our previous data concerning characteristics and the requirements of in vivo activation of alloantigen-specific DTH reactive T cells and of alloantigen-specific Ts cells suggest that different Ts cell populations are involved in suppression of alloantigen-specific DTH in vivo and of allogeneic suppression of in vitro induced sheep erythrocytes specific antibody formation.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Inactivation of the proteolytic activity of mouse nerve growth factor by human C1(activated)-inhibitor

The interaction between the serine protease gamma subunit of NGF (gamma-NGF) and human C1(activated)-inhibitor (C1-Inh) has been studied. C1-Inh inactivates the protease activity of gamma-NGF as measured by its ability to cleave the synthetic substrate benzoyl-arginine-p-nitroanilide (L-BAPNA). Experiments in which gamma-NGF and C1-Inh were mixed at differing molar ratios indicated that inhibition was due to the formation of a 1:1 stoichiometric complex. Analysis of the interaction of 125I-labeled gamma-NGF with C1-Inh by SDS-PAGE and autoradiography indicated that a covalent bond was formed between gamma-NGF and C1-Inh. The covalent bond was hydrolyzed by hydroxylamine, which suggested that the two proteins were linked via an acyl linkage. The formation of this complex was time dependent and required the proteolytic activity of the gamma-NGF.     

The sesquiterpene lactone hymenoxon acts as a bifunctional alkylating agent

Hymenoxon, a toxic sesquiterpene lactone found in bitterweed, bound deoxyguanosine in a cell free system and formed adducts with guanine residues in cellular DNA. The reactive dialdehyde form of hymenoxon formed stable Schiff base products with deoxyguanosine which were separable from unreacted hymenoxon and deoxynucleosides by reverse phase high pressure liquid chromatography. Hymenoxon adducts which eluted as a single impure peak from the octadecylsilane column separated on amino and diphenyl-bonded phases with 10% methanol. Tritiated nucleoside adducts were isolated and purified from CFW mouse sarcoma cells treated with hymenoxon. Proton nuclear magnetic resonance spectra of purified hymenoxon-deoxyguanosine adducts revealed a loss of signals for hydroxyl groups in the bishemiacetal of hymenoxon. ¹³C-nuclear magnetic resonance spectra revealed that the major adduct has 35 carbon atoms, indicating an interaction of at least two guanine residues per hymenoxon molecule and suggesting that hymenoxon may cross-link DNA. Sedimentation analysis of treated DNA further showed that DNA cross-linking by hymenoxon (30 µg/ml) was equivalent to that of a known cross-linking agent, mitomycin C (7.5 µg/ml). Hymenoxon was more cytotoxic to DNA cross-link repair-deficient Chinese hamster ovary cell mutants than to repair proficient strains. These data combine to indicate that hymenoxon acts as a bifunctional alkylating agent which cross-links DNA in mammalian cells.CHO Chinese hamster ovary - HYM hymenoxon - MMC mitomycin C - NMR nuclear magnetic resonance - PBS phosphate buffered saline     

Repeated sequence elements in the high molecular weight basic nuclear proteins from the winter flounder

In maturing sperm of the winter flounder, histones are not replaced by protamines but instead joined by a group of high molecular weight basic nuclear proteins. Despite their large size and number of components, these proteins were reduced to a relatively simple set of peptides by a "limit" digestion with endoprotease Lys-C. Nine of these peptides, that together account for half of the mass of the digest, were purified by two rounds of chromatography on a C18 reverse-phase high pressure liquid chromatographic column and analysed by sequential Edman degradation. Their sequences can be divided into two homology groups. Seven of the peptides contain all or part of a dodecapeptide consensus sequence, NH2-Ser-Pro-Met-Arg-Ser-Arg-Ser-Pro-Ser-Arg-Ser-Lys-COOH, which appears to be tandemly repeated. This dodecapeptide contains a previously recognized consensus phosphorylation sequence, NH2-Arg-Ser-Arg-Ser-Pro-COOH, in which both serines are phosphorylated during the early stages of spermiogenesis. The other homology group has the sequence NH2-Arg-Arg-Val-X-X-Pro-Lys-COOH, where X-X is either Gln-Thr or Pro-Ser. The dodecapeptide and heptapeptide sequences form at least 35 and 11%, respectively, of the high molecular weight basic nuclear proteins and are, therefore, repeated many times over in these proteins. A search for identical or homologous sequences within the Protein Sequence Database indicated that they are unique. The closest matches were to protamines and some viral DNA-binding proteins.     

Regional mapping panel for human chromosome 17: Application to neurofibromatosis type 1

A somatic cell hybrid mapping panel was constructed to localize cloned DNA sequences to any of 15 potentially different regions of human chromosome 17. Relatively high-resolution mapping is possible for 50% of the chromosome length in which 12 breakpoints are distributed over approximately 45 megabases, with an average spacing estimated at 1 breakpoint every 2-7 megabases. This high-resolution capability includes the pericentromeric region of 17 to which von Recklinghausen neurofibromatosis (NF1) has recently been mapped. Using 20 cloned genes and anonymous probes, we have tested the expected order and location of panel breakpoints and confirmed, refined, or corrected the regional assignment of several cloned genes and anonymous probes. Four markers with varying degrees of linkage to NF1 have been physically localized and ordered by the panel: the loosely linked markers myosin heavy chain 2 (25 cM) to p12----13.105 and nerve growth factor receptor (14 cM) to q21.1----q23; the more closely linked pABL10-41 (D17S71, 5 cM) to p11.2; and the tightly linked pHHH202 (D17S33) to q11.2-q12. Thus, physical mapping of linked markers confirms a pericentromeric location of NF1 and, along with other data, suggests the most likely localization is proximal 17q.     

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli.

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.     

Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.