Structural diversity among anti-p-azophenylarsonate monoclonal antibodies from A/J mice: Comparison of Id and Id sequences

Amino acid sequence analyses were carried out on monoclonal anti-p-azophenylarsonate antibodies isolated from the ascites of mice carrying cell lines obtained from the fusion of A/J splenic lymphocytes with the myeloma cell line Sp2/0–Ag14. The partial primary structures of both heavy and light chains from seven idiotype negative hybridoma proteins are compared to those of six idiotype positive molecules. Amino-terminal amino acid sequences (40–47 residues) of heavy chains from molecules bearing the major cross-reacting idiotype, Id^CR, demonstrated 95% homology to each other. Similarly, aminoterminal sequences of Id^CR+light chains were homologous to each other. However, sequence variations were evident in individual antibodies in both framework and complementarity-determining regions, suggesting that a large family of molecules accounts for the major cross-reacting idiotype, as previously reported (Marshak-Rothstein et al., 1980b).

Heavy and light chains from seven Id^CR-negative monoclonal antibodies were subjected to amino-terminal (37–48 residues) amino acid sequence analysis. Four heavy chains were blocked to Edman degradation, but could be sequenced after enzymatic removal of the amino-terminal pyrrolidone carboxylic acid residue. In comparison with Id^CR-positive heavy chains, the Id^CR-negative heavy chains demonstrate greater diversity in both framework and complementarity-determining regions, with several different subgroups represented in contrast to the results from pooled serum Id^CR-positive antibodies (Capra et al., 1975). One of the seven Id^CR-negative light chains was blocked. The sequences of the remaining Id^CR-negative light chains exhibited marked variations in both framework and complementarity-determining regions, with different chain lengths in the first complementarity-determining region in several light chains.

Comparisons between the amino-terminal sequences of Id^CR-positive and Id^CR-negative monoclonal antibodies suggest that specific sequences in the first complementarity-determining regions of both heavy and light chains are not sufficient to account for the major cross-reacting idiotype. The structural basis for Id^CR in A/J mice is likely to be in other segments of the variable regions.     

Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3 coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5 end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.     

An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies to different parts of the gram-negative lipopolysaccharide core region

Bovine serum albumin was complexed with the core antigens of either Escherichia coli J5 LPS, Salmonella minnesota R595 LPS or E. coli lipid A. These core-BSA complexes were used for solid-phase coating in ELISAs for anti-core antibodies. Antibodies, binding to various parts of the core region were easily quantified in a single experimental set-up, which was hitherto not possible. The ELISA has only 3 incubation steps and is not costly as only moderate amounts of the core antigens (i.e., 1 microgram per test) were needed for coating. The sensitivity proved to be excellent and the complexes were biologically fully active (compared to native, smooth LPS), which make them suitable for the screening (after fusion) of monoclonal anti-core antibodies. Another possible application is the large-scale screening of blood-bank sera in order to find samples with a high anti-core antibody content.     

增殖细胞核抗原表达与AgNORs图像分析在判断肺癌预后上的应用

应用抗增殖细胞核抗原（抗ＰＣＮＡ）单克隆抗体，采用Ｓ－Ｐ免疫组化方法和ＡｇＮＯＲｓ染色图像分析，对有确切随访结果的８６例原发性肺癌和１０例肺炎性假瘤进行了研究。结果表明：经ＰＣ－ＮＡ阳性反应计算出的增殖指数（ＰＩ）与图像分析的ＡｇＮＯＲｓ计数及其颗粒总面积间存在着明显的相关性。二者均与肺癌分型有关，并随癌分化程度的增高而减小；与ＴＮＭ分期中的淋巴结（Ｎ）和远隔转移（Ｍ）有关；术后存活５年以上者明显小于３年以内者（Ｐ＜０．０１）。认为ＰＣＮＡ的表达和ＡｇＮＯＲｓ图像分析技术有助于肺癌的分型、分化及预后的判断。     

抗人C1—抑制物单链抗体的构建

由一株抗人Ｃ１－抑物单克隆抗体鼠杂交瘤细胞提取总ＲＮＡ，合成２对与免疫蛋白可变区ＦＲ１和ＦＲ４互补的通用引物，经ＲＴ－ＰＣＲ扩增出抗体的重链可变区和轻链可变区基因。     

Functional mapping of the activity of the R region in the human T-cell leukemia virus type I long terminal repeat to increase gene expression

We previously demonstrated the activity of the R fragment in the long terminal repeat of human T-cell leukemia virus type I for elevation of the level of gene expression. In this study, the fragment was deleted with BAL31 nuclease to determine its functional domain. Series of the shortened R fragments were linked to the simian virus 40 promoter unit, which regulated expression of a reporter gene. Examination with the R fragments deleted from the 5 and 3 ends showed that borders of the functional domain were mapped within nucleotide positions 458 to 473 for the 5 end and nucleotide positions 559 to 594 for the 3 end, respectively. Thus we conclude that a 136-base-pair fragment corresponding to the second half of the R region was sufficient to allow elevation of the level of gene expression.     

Subtle translocation (18;21) confirmed by FISH in a patient with Down syndrome

The patient presented with the typical features of Down syndrome: hypotonia, brachycephaly, flattened occiput, bilateral prominent medical epican-thic folds, flat nasal bridge, protruding tongue, low-set dysplastic ears, short broad hands, bilateral clinodactyly and simian crease. The karyotype of this child was originally reported as normal. High-resolution chromosomes revealed extra material on the long arm of chromosome 18. The mother's karyotype showed a reciprocal translocation between the long arm of 18 and the long arm of 21 at band q23 and q22.1, respectively. FISH performed separately with two different 21q cosmid probes gave two signals on the mother's metaphases and three signals on the prob-and. These findings confirmed that the proband is trisomic for the long arm of chromosome 21 at loci D21S65 and D21S19.     

Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor

Characterization of the human placental membrane receptor for human ¹²⁵I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10⁷ mole^?1, and 2.0 ± 0.16 × 10¹⁵ IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 mM), and periodate (4 mM) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 mM or 20 mM) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of ¹²⁵I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.     

Ribosomal DNA internal transcribed spacer sequences do not support the species status of Ampelomyces quisqualis, a hyperparasite of powdery mildew fungi

Phylogenetic relationships among Ampelomyces isolates, pycnidial hyperparasites and biological control agents of powdery mildews, were inferred from internal transcribed spacer (ITS) sequences of the ribosomal DNA (rDNA). Currently, these hyperparasites are considered to be a single species, A. quisqualis, despite observed morphological and cultural differences. Ten Ampelomyces isolates, representing seven previously defined ITS RFLP groups, were sequenced and analyzed. Sequence-divergence values among isolates belonging to different RFLP groups ranged from 4.3 to 22.4%, suggesting that these isolates may represent different taxa. When Ampelomyces ITS sequences were analyzed by cladistic methods with the sequences of other ascomycetous fungi, they formed two lineages in the Dothideales. Slow-growing Ampelomyces isolates formed a clade with Leptosphaeria microscopica and L. nodorum, whereas fast-growing Ampelomyces isolates formed a clade with Epicoccum nigrum. Sequence-divergence values between these two clades ranged from 17.3 to 22.4%, suggesting that the taxa in the two clades are not closely related and possibly not congeneric. The data presented here indicate that the identification of `A. quisqualis' isolates used in biological control experiments should be re-evaluated. Received: 10 March 1997 / Accepted: 13 February 1998