Degeneracy of antibody specificity

Evidence was obtained, by direct binding assays (radioimmunoassays) and by inhibition of binding assays, that after immunization some of the antibody molecules produced are degenerate in that they bind not only the immunizing antigen, but also unrelated ligands. It can be concluded that the exquisite specificity of the immune response is not necessarily a property of any given antibody molecule but is, at least to some extent, due to the summation of specificities held in common by the population of antibody molecules produced during the response.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [14C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2'-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined in cell extracts. The results demonstrate that under physiological conditions, there is a small but significant flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5'-nucleotidase (EC 3.1.3.5), reflecting the markedly higher Vmax/Km ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher Vmax/Km ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts

Injection of Fanconi anaemia (complementation group A) fibroblasts with HeLa mRNA is shown to correct their abnormal response to a psoralen cross-linking challenge, namely permanent repression of DNA synthesis. Injection of gradient-fractionated mRNA led to identification of a single fraction, containing mRNA of approximately 650 bases, which is responsible for this effect. This finding suggests that Fanconi anaemia (group A) cells are deficient in a small protein, up to 20 kDa in size, which is involved in the cellular response to DNA interstrand cross-links.     

Ultrastructural immuno-localization of tropoelastin in the chick eye

Summary Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.     

Fanconi's anaemia: correlation of genetic complementation group with psoralen/UVA response

Summary The correlation found by Moustacchi (1987) between cellular response to a crosslinking challenge and genetic heterogeneity in Fanconi's anaemia is confirmed for an earlier set of complementation groups (Zakrzewski and Sperling 1980). This allows the matching of the two independently established complementation groupings and better characterization of their DNA repair-related biochemical properties.