Monoclonal antibodies to antigens abnormally expressed in breast cancer

We report the production, screening, and characterization of ten murine monoclonal antibodies directed at antigens that are expressed abnormally in human breast tumors. Immunoperoxidase staining of frozen and fixed tissues shows the antigens to be present at low levels on the luminal membrane of normal breast cells and at high levels in the cytoplasm and surface membrane of breast tumor cells. The ten antibodies appear to recognize six different epitopes on the basis of their quantitative differences in reactivity against four antigen preparations, as measured by ELISA. Immunoblots show that eight of the ten antibodies recognize a 300,000 MW molecule from breast tumor preparations; six of these antibodies also react with a second molecule from the same tumor preparations of 280,000 MW. Seven antibodies react with an antigen from milk fat globule membrane of 330,000 MW. It therefore appears that the two molecules from tumor tissue and the one molecule from normal tissue share common epitopes. Selected antibodies were tested for reactivity against 25 primary breast tumors and 14 pairs of primary and metastatic breast tumors. Three antibodies have broad reactivity and stain more than 80% of primary tumors; the three other antibodies identify subsets of those tumors. Results of staining pairs of primary and metastatic lesions show that metastases continue to express antigens of the primary lesion in a high percentage of cells.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.     

Histochemical and ultrastructural data on the adrenergic and cholinergic innervation of the epididymis in the mouse

The adrenergic and cholinergic innervation of the epididymal duct in the mouse has been investigated using histochemical methods and electron microscopy. This study demonstrates that the epididymal innervation of the mouse does not really differ from that of other mammals. Cholinergic nerves are mainly vascular, even in the cauda where cholinesterase activity is increased within the tubular musculature. Catecholamine fibres ensure the motricity of the wall of the canalicular system, particularly the terminal segment where the smooth muscle fibres are specifically differentiated.     

Meiofauna associated with a Pacific coral reef in Costa Rica

The meiofauna of two coral reef habitats at Isla del Naño, Costa Rica was studied over a one year period. The dominant groups were: Foraminifera (21.2%), Copepoda (19.7%), Nematoda (19.1%), Gastropoda (16.5%), Polychaeta (7.2%) and Bivalvia (6.6%). The highest diversity was found in coarse, heterogeneous sands with the highest percentage of carbonates. The meiofauna showed a high degree of horizontal aggregation, which is a characteristic pattern for macro- and meiofauna in sediments of variable composition. No vertical variation in distribution was evident, probably due to the deep location of the Redox Potential Discontinuity layer. The total densities of organisms found in this study (99 to 575 ind/10 cm²) are low compared with densities in similar non-reefal sands (7 to 6116), and from fine sediments (80 to 17 000), but are comparable to densities found in other reef areas (39 to 609.5 ind/10 cm²). This is the first report on meiofauna from the eastern Pacific, and the first time that foraminiferans are the dominant group.     

Molecular diagnosis of Duchenne and Becker muscular dystrophies. Current data

Carrier diagnosis and prenatal diagnosis of Duchenne's muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD) has become possible using some twenty RFLPs detected by more than a dozen Xp21 probes that are either intragenic or flanking the disease locus. Results from familial studies on 88 DMD and BM families stress important considerations concerning a priori and final risks, individuals necessary for the identification of the phase, and the different strategies that can be applied, regardless of whether the study concerns an on-going pregnancy or a carrier-status determination, and whether the patient is at high or low risk. Finally, multiple sources of difficulties in interpreting the results depend on a) the occurrence of new mutations that must be traced; b) the existence of meiotic recombination; c) the necessity, in some instances, of relying upon the sole identification of the paternal X. These considerations emphasize the characteristics and the important limitations of this type of methodology.     

Conformational predictive studies on the activation segment of pancreatic procarboxypeptidases

Little is known about the conformation and evolutionary origin of the activation segment of pancreatic procarboxypeptidases. Analysis of the sequence and secondary structure propensities of these propeptide segments indicate that they contain two regions structurally related to the Ca2+-binding sites of the EF-hand protein family. This proposed homology could explain how (and why) carboxypeptidases developed such long (94 residues) activation peptides.     

Microheterogeneity of the malate dehydrogenase from several sources

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.     

trans-Diamminedichloroplatinum(II), a reversible RNA-protein cross-linking agent. Application to the ribosome and to an aminoacyl-tRNA synthetase/tRNA complex

A new approach allowing detection of contact points between RNAs and proteins has been developed using trans-diamminedichloroplatinum(II) as the cross-linking reagent. The advantage of the method relies on the fact that the coordination bonds between platinum and the potential acceptors on proteins and nucleic acids (mainly S of cysteine or methionine residues; N of imidazole rings in histidine residues; N7 of guanine, N1 of adenine, and N3 of cytosine residues) can be reversed, so that the cross-linked oligonucleotides or peptides in contact within a complex can be analyzed directly. The method was worked out with the ribosome from Escherichia coli and the tRNAVal/valyl-tRNA synthetase system from the yeast Saccharomyces cerevisiae. In the first system the platinum approach permitted detection of ribosomal proteins cross-linked to 16S rRNA within the 30S subunits (mainly S18 and to a lower extent S3, S4, S11, and S13/S14); in the second system major oligonucleotides of tRNAVal cross-linked to valyl-tRNA synthetase were detected in the anticodon stem and loop, in the variable loop, and in the 3' terminal amino acid accepting region. These results are discussed in light of the current knowledge on ribosome and tRNAs and of potential applications of the methodology.     

A new relationship between cholesterolemia and cholesterol synthesis determined in rats fed an excess of cystine

The present study deals with an attempt to describe how the plasma cholesterol level is related to input into the plasma of cholesterol synthesized in the liver and in the intestine. It has previously been shown in our laboratory that, for a given absorption of alimentary cholesterol, the rat plasma cholesterol level decreases when internal secretion of cholesterol (cholesterol synthesized in the organs and poured into the plasma) increases. This relationship was established using rats in which the major source of cholesterol synthesis was the intestine. We used rats fed a cystine-enriched diet (5%) which was previously shown to increase cholesterolemia and internal secretion of cholesterol. It was first demonstrated that a significant positive linear correlation exists between individual values of cholesterolemia and those of internal secretion of cholesterol. Secondly, using [14C]acetate as the cholesterol precursor it was shown that ingestion of the cystine-enriched diet increased hepatic but not intestinal cholesterogenesis. Individual values of cholesterolemia were linearly correlated to those of [14C]acetate incorporation into the hepatic sterols. Results obtained by this method were validated by determining the 13C-labeling pattern of cholesterol synthesized de novo by the liver and the intestine after [13C]acetate infusion. Indeed, this labelling indicated that the dilution of exogenous acetyl-CoA in the liver was not changed by cystine feeding, whereas that in the intestine was enhanced. It is concluded that the plasma cholesterol level varies with internal cholesterol secretion, depending on the organ which determines the variations of this secretion: it decreases when intestinal cholesterogenesis increases, whereas it increases when hepatic cholesterogenesis increases. Finally, the use of [14C]acetate coupled with lipoprotein analysis in rats fed the cystine-enriched diet, in control rats and in rats fed a cholesterol-enriched diet, allowed a new linear correlation to be demonstrated: between cholesterol concentration in LDL2 (lipoproteins of density 1.040-1.063 g/ml) and [14C]acetate incorporation into liver sterols. Our results suggest that LDL2 are produced by the liver in relation to cholesterogenesis in this organ.     

Availability to monoclonal antibodies of antigenic sites of the alpha and beta subunits in active, denatured or membrane-bound mitochondrial F1-ATPase

The binding of five monoclonal antibodies to mitochondrial F1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F1 integrated in the mitochondrial membrane, for purified beta-subunit and for purified F1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-beta monoclonal antibodies bound more easily to the beta-subunit than to active F1, and recognized equally active F1 and F1 integrated in the membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated beta-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-beta monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the beta-subunit. One anti-alpha monoclonal antibody (20 D6) bound poorly to F1 integrated in the membrane, while the other (7 B3) had a higher affinity for F1 integrated in the membrane than for soluble F1. Therefore, 20 D6 recognizes an epitope of the alpha-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the alpha-subunit well exposed at the surface of the inner face of the mitochondrial membrane.