Unique B-1 cells specific for both N-pyrrolated proteins and DNA evolve with apolipoprotein E deficiency

Lysine N-pyrrolation, a posttranslational modification, which converts lysine residues to N^ε-pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE^−/−) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE^−/− mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE^−/− mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.     

A novel family of variable region genes of the human immunoglobulin heavy chain

We isolated and sequenced six variable-region (V) gene segments of the human immunoglobulin heavy-chain (H) using the V71-2 segment as probe. These VH segments were more than 90% homologous to each other and less than 65% homologous to members of the three known VH families. The VH fragments hybridized to an identical set of restriction fragments on Southern blots of human placenta DNA. The new family was designated as the VH-IV family. The complexity of the VH-IV family was estimated to be at least nine genes, of which the sequenced seven were functional genes. The VH-IV family is homologous (76%) to the mouse Vh36-60 family.     

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity

Hybrid peptides were constructed from endothelin B receptor (ET_B) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ET_B as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ET_B but for ET_A. Although all analogues had antagonistic effects on ET_A, some analogues had proved to function as agonist on ET_B confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ET_B-selective agonist with weak ET_A antagonism (3, KT7421); (2) ET_B-selective antagonist with weak ET_A antagonism (29, KT7539); (3) ET_B agonist with potent ET_A antagonism (27, KT7538); and (4) non-selective ET_A/ET_B antagonist (26, KT7540).     

Role of chloroplast trienoic fatty acids in plant disease defense responses

Trienoic fatty acids (TAs) are the major polyunsaturated fatty acid species in the membrane lipids in plant cells. TAs are crucial for the adaptation to abiotic stresses, especially low- or high-temperature stress. We show that TAs in chloroplast membrane lipids are involved in defense responses against avirulent bacterial pathogens. Avirulent pathogen invasion of plants induces a transient production of reactive oxygen intermediates (ROI), programmed cell death and subsequent disease resistance. The Arabidopsis fad7fad8 mutation, which prevents the synthesis of TAs in chloroplast lipids, caused the reduction in ROI accumulation in leaves inoculated with Pseudomonas syringae pv. tomato DC3000 (avrRpm1). Linolenic acid, the most abundant TA, activated the NADPH oxidase that is responsible for ROI generation. TAs were transferred from chloroplast lipids to extrachloroplast lipids coincident with ROI accumulation after inoculation with Pst DC3000 (avrRpm1). Furthermore, the fad7fad8 mutant exhibited reduced cell death and was compromised in its resistance to several avirulent P. syringae strains. These results suggest that TAs derived from chloroplast lipids play an important role in the regulation of plant defense responses.     

Structure of the human ornithine transcarbamylase gene

Complementary and genomic DNA clones corresponding to the human ornithine transcarbamylase (OTC) [EC 2.1.3.3]mRNA have been isolated and analyzed. The OTC gene is about 73 kilobase pairs (kb) long and contains 10 exons interrupted by 9 introns of highly variable sizes. The smallest intron is 80 base pairs and the largest, 21.7 kb. The 5'- and 3'-flanking regions, entire exons and all the exon/intron boundaries were sequenced. The nucleotide and deduced amino acid sequences of isolated OTC cDNAs as well as the corresponding regions of the genomic DNA were compared with those of human OTC cDNA (Horwich, A.L., Fenton, W.A., Williams, K.R., Kalousek, F., Kraus, J.P., Doolittle, R.F., Koningsberg, W., & Rosenberg, L.E. (1984) Science 224, 1068-1074). We found 20 nucleotide substitutions among these sequences, of which 6 related to amino acid changes. The nature of these nucleotide substitutions is discussed.     

Open reading frame vpr of simian immunodeficiency virus encodes a virion-associated protein.

The genomes of simian immunodeficiency viruses isolated from rhesus macaques (SIVmac) contain an open reading frame (ORF), vpr, which has a coding potential of 97 to 101 amino acid residues. In this study, a vpr ORF-encoded protein of approximately 11 kDa was identified, and anti-vpr antibodies were detected in rhesus macaques infected by SIVmac. These results provide clear evidence that the vpr ORF is a coding gene of SIVmac. The vpr protein, like the vpx protein which is encoded by another accessory gene of SIVmac, was also found to be associated with viral particles. This observation demonstrates that more than one accessory gene product can be present in the virions of this family of retroviruses and raises the possibility that the vpr protein may have a role in early part of the virus life cycle.