A phosphorylcholine idiotype related to TEPC 15 in mice infected with Ascaris suum.

A serum component, binding antigens having phosphorylcholine (PC) determinants were induced in several strains of mice by infection with Ascaris suum. This component was isolated and demonstrated to be an IgM (K) anti-PC antibody having idiotypic determinants in common with the IgA PC-binding myeloma protein TEPC 15. Rabbit anti-idiotypic antisera prepared with this component had idiotypic specificity for TEPC 15 and cross-idiotypic recognition of MOPC 167 and McPC 603, all IgA PC-binding myeloma proteins. The antisera also recognized determinants not present on TEPC 15. IgM and idiotype levels were quantitated by radial immunodiffusion and PC-specific antibody measured by hemagglutination (HA) with sheep erythrocytes coated with pneumococcal-C-polysaccharide. Mean IgM levels ranged from 2.5 to 8.7 mg/ml, idiotype from 0.54 to 5.3 mg/ml; and HA titers from 1:512 to 1:130,000 in different mouse strains. The high PC-specific antibody response was not duplicated by immunization with dead ascaris larvae or by infection with two other nematode species.     

Repertoire diversity of antibody response to bacterial antigens in aged mice. II. Phosphorylcholine-antibody in young and aged mice differ in both VH/VL gene repertoire and in specificity

Aging of mice is accompanied by both quantitative and qualitative changes in antibody responses to phosphorylcholine (PC), an immunodominant epitope of Streptococcus pneumoniae R36a strain (Pn). In order to study these changes at the molecular level, we generated PC-specific hybridomas from young (3 to 4 mo) and aged (20 to 24 mo) mice of different strains after primary immunization with S. pneumoniae R36a strain. These mAb were tested for Ig VH and VL gene family utilization, idiotopic repertoire, and cross-reactivity with unrelated Ag. Hybridomas from young mice (BALB/c, C57BL/6, and D1.LP) uniformly expressed the VH-S107 and V kappa-22 genes as well as most idiotopes of the T15 family, which were identified with different anti-T15 mAb. In contrast, the PC-reactive mAb from aged mice were quite heterogeneous: only 2 out of 13 utilized VHS107, 1 of 13 used VH7183, and 3 of 13 used VHJ558 gene family. Moreover, none of these mAb used L chain encoded by V kappa 22(0/13), but surprisingly they frequently expressed some of the T15 idiotope. In addition, the PC-binding mAb from aged mice showed broad cross-reactivity with various mouse and foreign proteins, whereas the mAb from young mice did not. These results demonstrate the genetic shift in antibody response of aging mice to PC, which is accompanied by a change in the antibody specificity. Interestingly, the qualitative repertoire change appears to be unrelated to the magnitude of antibody response, for the aged BALB/c mice maintain a very high reactivity to PC.     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

Altered VH gene segment utilization in the response to phosphorylcholine by aged mice

Newly generated bone marrow B cell precursors of aged BALB/c mice, stimulated in splenic fragment cultures, display a markedly increased frequency of phosphorylcholine (PC)-responsive cells. This increased frequency is found for both precursors that utilize VHS107, a phenotype common to essentially all PC-specific B cells of young mice, and, surprisingly, for precursors that utilize VH genes other than VHS107. PC-specific hybridomas derived from bone marrow cells of aged mice utilize members of at least three VH gene segment families that have never been observed in PC responses of young mice. The ability of aged but not young mice to generate these unique PC-specific clonotypes may be evidence for constraints on V region utilization during repertoire development in young adults and has important implications for aging-associated changes in immune responsiveness.     

Binding of phosphorylcholine by non-immunoglobulin molecules on mouse B cells

Phosphorylcholine (PC), a molecule found in the cell wall of most serotypes of pneumococcus, has been used extensively as a probe for the study of network interactions during immune responses. The frequency of B lymphocytes capable of interacting with PC has not been directly examined. We used immunofluorescence to study the binding of PC and monoclonal anti-TEPC15 anti-idiotopic antibodies to murine lymphocytes. In addition to identifying PC-specific Ig molecules, PC was bound by a non-Ig molecule on the surface of a relatively large subset of B cells; this non-Ig marker shared an idiotypic determinant with the PC-binding myeloma protein HOPC8 (H8). PC-bearing R36a pneumococci bind to a similar subset of lymphocytes. This binding is inhibited specifically by PC coupled to bovine serum albumin and also by a monoclonal anti-H8 antibody. We suggest that bacterial interaction with B cells through non-Ig molecules capable of binding a dominant antigen like PC may possess functional significance, possibly during the events that lead to antibody induction by these microorganisms.     

T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of T15i knockin mice.

T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the J(H) region of chromosome 12. This T15H chain combines with a kappa22-33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id-. Analysis of L chains expressed in these T15-Id-, PC-specific B cells revealed that two L chains, kappa8-28 and kappa19-15, were expressed in this population. The V(kappa) region of these L chains was recombined to J(kappa)5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id- B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id- B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:kappa8-28L and T15H:kappa19-15L Abs revealed L chain differences from the kappa22-33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:kappa22-33L Ab.     

Biochemical properties of phosphatidylcholine-binding proteins that share common antigenic determinants with Fc gamma 2b receptor

Biochemical and immunological properties of biosynthetically radiolabeled phosphatidylcholine-(PC-) binding proteins were investigated. The PC-binding proteins were extracted from the detergent lysate of biosynthetically radiolabeled P388D1 cells by affinity chromatography on PC-Sepharose and filtered through a Sephadex G-100 gel column in the presence of 6 M urea. Isoelectric focusing of the gel-filtered materials in the presence of 6 M urea revealed the presence of a major protein component of pIe of 5.8 and minor heterogeneous cellular proteins. The yield of the electrofocused PC-binding proteins based on protein determination by Lowry's method ranged from 0.7 to 4 mg per 10(9) cells. The purified PC-binding proteins appeared to be tightly associated with Triton X-100 and phospholipids in the weight ratio of 0.57 and 0.05 g/g of proteins, respectively. The majority of lipids that could be extracted from the PC-binding proteins by chloroform/methanol (2:1 v/v) are free fatty acids, whereas lipids extracted from Pronase-treated PC-binding proteins contained phosphatidylethanolamine. By amino acid analysis, the purified PC-binding proteins were found to consist of a minimum of 417 amino acid residues, suggesting a minimum molecular weight of about 38 000 for this protein. Results of radiolabeling experiments with [3H]glucosamine and amino acid analysis both showed the presence of a mole of glucosamine per a mole of the PC-binding proteins, suggesting their glycoprotein nature. About 40% of the purified PC-binding proteins coprecipitated with monoclonal anti-Fc gamma 2bR antibody (2.4G2) in detergent-containing buffer, whereas only 6% of the isolated IgG binding proteins reacted with this antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoreactive B cells escape clonal deletion by expressing multiple antigen receptors

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2-/-) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2-/- knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.     

T15 D region germ line amino acid sequences distinguished by monoclonal anti-idiotope antibody

Monoclonal antibody NL16, prepared with phosphorylcholine (PC)-binding myeloma protein C.BBPC3 (C3), identified an idiotope (C3-16 Id) that was present on T15 IdX+ myeloma proteins (MP) C3, T15, and H8, but not the T15 IdX- MP M167 and M603. The binding of C3 to NL16 is PC inhibitable, indicating that C3-16 Id is site associated. Inhibition studies with PC-specific hybridoma proteins (HP) demonstrated that the T15-type L chain VK22 and elements of the H chain were required for C3-16 Id expression. Studies of amino acid sequences of these PC-binding HP and MP showed that VK22+, T15 IdX+ HP, and MP that use the T15 D region (YYGSS) sequences were always C3-16 Id+. However, the reverse was not true, because all but one VK22+, T15 IdX+ HP with D region sequence changes were C3-16 Id-. This suggested that NL16 defined a specificity mainly determined by the D region of the H chain. A direct test of this hypothesis with heterologous heavy/light chain recombinant molecules obtained from C3-16 Id+ and C3-16 Id- HP of known sequence, showed that the D region was critical to idiotope expression. Additionally, an examination of the amino acid sequences of VK22+, T15 IdX- HP, HPCG14, and HPCM6 suggest that profound changes in the D region may also alter the expression of T15 IdX (an Id defined by a multispecific antiserum from A/He mice). The C3-16 Id+ was found in anti-PC serum of most Ig haplotype-inbred strains except for CBA/J, C3H, and PL, which are all of the Igh-Cj haplotype. Amino acid sequences of PC-binding CBA and PL HP showed marked changes in the D region from the T15 type, and this may account for the C3-16 Id- character of Igh-Cj strains.     

Limunectin. A phosphocholine-binding protein from Limulus amebocytes with adhesion-promoting properties

A protein that binds to and precipitates with pneumococcal C-polysaccharide and a phosphocholine (PC) derivative of bovine serum albumin has been affinity purified from Limulus amebocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals that the isolated protein consists of a single polypeptide chain of approximately 50 kDa. It is an intracellular protein localized in the secretory granules of amebocytes according to immunogold staining. Although it shares the PC-binding property with C-reactive protein isolated from Limulus and other animal species, it differs from C-reactive protein in that the latter binds to PC only in the presence of Ca2+, whereas the newly isolated protein binds to PC in a Ca(2+)-independent manner. In this respect, the newly isolated PC-binding protein resembles the antibodies to PC of mouse myelomas. The gene coding for this protein has been isolated. The gene sequence predicts a protein of 54 kDa with an unusual structural feature: it consists almost entirely of 10 contiguous segments, 45 amino acids in length, with extensive homology. Some limited sequence homologies were found between the 54-kDa protein and segments of vitronectin, gelatinase, and collagenase. It binds to bacterial cells, fixed amebocytes, and a number of extracellular matrix molecules. Due to its structural and some functional similarities to other adhesion molecules, the Limulus 54-kDa protein was named "Limunectin."     

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.     

Diversity of carbazole-degrading bacteria having the car gene cluster: isolation of a novel gram-positive carbazole-degrading bacterium

Twenty-seven carbazole-utilizing bacterial strains were isolated from environmental samples, and were classified into 14 groups by amplified ribosomal DNA restriction analysis. Southern hybridization analyses showed that 3 and 17 isolates possessed the car gene homologs of Pseudomonas resinovorans CA10 and Sphingomonas sp. strain KA1, respectively. Of the 17 isolates, 2 isolates also have the homolog of the carAa gene of Sphingomonas sp. strain CB3. While the genome of one isolate, a Gram-positive Nocardioides sp. strain IC177, showed no hybridization to any car gene probes, PCR and sequence analyses indicated that strain IC177 had tandemly linked carAa and carC gene homologs whose deduced amino acid sequences showed 51% and 36% identities with those of strain KA1.     

Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.     

Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus

Pandemic V. parahaemolyticus strains have rapidly changed their serotypes, but its determinants, especially K antigen, and the genes involved in serotype have been an open question. The purpose of this study was to gain insights into these points. Although V. parahaemolyticus is known to be lacking O-side chain on its lipopolysaccharide, and O antigens are thought to be represented by core OS, the genome sequence of V. parahaemolyticus O3:K6 strain RIMD2210633 suggests that this bacterium potentially synthesizes O-side chain. To explore possible relatedness between this O-side chain biosynthesis gene cluster, which is similar in the serotypes of Vibrio cholerae, and of V. parahaemolyticus, we amplified both core OS and O-side chain gene clusters of the strains belonging to various serotypes of V. parahaemolyticus by long PCR and performed PCR RFLP analyses. The results of our RFLP analyses suggest that the core OS biosynthesis gene cluster is related to the O antigens of pandemic V. parahaemolyticus and that the putative O-side chain gene cluster is related to K antigens of pandemic V. parahaemolyticus. We then determined the sequence of these regions of a pandemic O4:K68 strain, and compared it with the corresponding sequence of RIMD2210633. In addition, PCR analysis showed the putative O4 and K68 antigen gene clusters are unique to the strains belonging to the O4 and K68 serotype respectively. The data implies that the pandemic O4:K68 V. parahaemolyticus strain emerged from the pandemic O3:K6 strain by replacement of the putative O and K antigen gene clusters.     

Activation of a stress-induced gene by insecticides in the midge, Chironomus yoshimatsui

Stress proteins (heat shock proteins, HSPs) have been proposed as general biomarkers for environmental monitoring. In the present study, we evaluated the environmental stress-burden on the aquatic midge Chironomus yoshimatsui using hsp70 expression. Larvae collected from streams receiving polluted runoff (field strain) were resistant to the organophosphorus insecticide, fenitrothion (F), and the synthetic pyrethroid, ethofenprox (E), whereas a strain originally collected from an unpolluted area (susceptible strain) showed low resistance to insecticide exposure. To examine the expression of an HSP70 gene in C. yoshimatsui, an hsp70 cDNA probe was prepared using RNA obtained from the field strain larvae and used for Northern blot analyses. The expression of this HSP70 gene in larvae collected from two field sites in May about 1 week after insecticide spraying in the fields was 2.3 (p = 0.018) to 3.3 fold higher than that in the susceptible strain and was also 4.6 and 1.4 (p = 0.033) fold higher than those collected in November 3 months after the cessation of insecticide spraying. In order to identify potential inducers of the HSP70 gene of the field strain, larvae of the susceptible strain were exposed to F or E for 24 h and hsp70 mRNA levels determined. Exposures to F at 0.4 microg/L and E at 1.1 microg/L increased hsp70 mRNA levels 2.7 (p = 0.049) and 4.4 (p = 0.043) fold over controls, respectively. These results suggest that larvae collected from polluted areas are burdened by environmental stressors and the tested insecticides are potential inducers of HSP70. The results also support the suggestion that HSP70 gene expression is a sensitive indicator of low level (nonlethal) exposures to certain insecticides.     

猪丹毒丝菌C43311株spaA基因N端免疫保护区的克隆和表达

采用PCR从猪丹毒丝菌C43311株基因组中扩增出编码表面保护性抗原A的spaA基因片段,将其克隆到pMD18-T载体并对插入片段进行测序.用spaA基因N端免疫保护区(spaA-N)的特异引物从重组质粒pMD18-spaA中扩增得到spaA-N片段,将其定向插入原核表达载体pGEX-6p-2中,构建重组表达质粒pGEX-spaA-N,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白GST-SpaA-N,用SDS-PAGE和Western印迹检测表达产物.测序结果表明spaA基因片段大小为1881bp,与已报道的不同血清型菌株spaA基因的核苷酸序列比较,核苷酸序列同源性在93%～99%之间.SDS-PAGE结果显示表达蛋白约为64kDa,与预期的大小相近.Western印迹检测结果表明,表达的融合蛋白GST-SpaA-N能与C43311株SpaA蛋白的抗血清发生特异性反应,证明原核融合表达蛋白具有免疫反应性.     

Transformation of Drosophila melanogaster with a suppressor tRNA gene (Sup3e tRNA(SerUGA)) from Schizosaccharomyces pombe

P-element mediated transformation was utilized to introduce a suppressor tRNA gene (Sup3e tRNA(UGASER)) from Schizosaccharomyces pombe into Drosophila melanogaster. Thirteen independently transformed lines were characterized as to the number of cytological locations of the transposons. It was ascertained that the suppressor tRNA gene of interest was introduced into each transformed strain. The helper P element used (p pi 25.1) allows further transposition to occur, and it was determined that from one to seven copies of the heterologous tRNA(UGASER) gene per strain were present among the respective transformed strains. The number of transposons per transformed line was established by in situ hybridization to salivary gland chromosomes as well as by Southern hybridization analyses and there was good agreement in the totals determined by these two techniques.     

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.     

Escherichia coli mutants defective in the uncH gene.

Plasmids carrying cloned segments of the unc operon of Escherichia coli have been used in genetic complementation analyses to identify three independent mutants defective in the uncH gene, which codes for the delta subunit of the ATP synthetase. Mutations in other unc genes have also been mapped by this technique. ATPase activity was present in extracts of the uncH mutants, but the enzyme was not as tightly bound to the membrane as it was in the parental strain. ATP-dependent membrane energization was absent in membranes isolated from the uncH mutants and could not be restored by adding normal F1 ATPase from the wild-type strain. F1 ATPase prepared from uncH mutants could not restore ATP-dependent membrane energization when added to wild-type membranes depleted of F1. Membranes of the uncH mutants were not rendered proton permeable as a result of washing with low-ionic-strength buffer.