Biomedical subjects
Publications and source records attributed to E Calef.
The Epstein-Barr virus genome contained in the Burkitt lymphoma line Namalwa was previously localized to the short arm of chromosome 1. Analysis of a different subline of the same Namalwa line by means of Southern analysis carried out on genomic DNA, as well as in situ hybridization, showed a localization on the X chromosome.
We show here that in a lymphoblastoid cell line Epstein-Barr virus DNA recombines with the human genome. The genetic exchange involves the oriP region of the virus. A junction between viral and human DNA from this line has been cloned and sequenced. The results indicate that the integration of Epstein-Barr virus DNA involves a region of the human genome which contains internal short repetition. An 800-bp probe has been isolated from the human part of the junction. This probe has been used to show that the human region exists as a duplication in normal cells.
Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.
Localization of Epstein-Barr virus (EBV) DNA was studied by in situ hybridization on chromosomes from the Namalwa Burkitt lymphoma cell line and from a lymphoblastoid cell line transformed in vitro (ATL9/g). The five chromosome bands 1p32, 1q31, 5q21, 13q21, and 16p13 showed the presence of EBV DNA in both of the lines. Grain deposition at the site on chromosome 1q of the Burkitt line was particularly intense. It was also found that EBV DNA in the lymphoblastoid cell line co-localized with a stable achromatic gap at 1p32 whose presence seems to confer a proliferative advantage on the cells.
Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This "non-plasmidic" pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements.
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Lethal recognition between Entamoeba histolytica and the tissues and defence systems of the host results in a continuous interplay that determines the development of pathological lesions: (i) we have identified several of the steps and mediators utilized by the trophozoites to destroy host cells by contact-mediated cytolysis; (ii) we have established that the alternative complement system represents the main defence available to the host against the invading parasite. The amoebae recognize target cells by means of a lectin specific for N-acetylgalactosamine-containing surface glycoproteins. This recognition appears to activate the amoeba to release, in the area of contact, an attack complex that induces the host cells to undergo cytolysis. The main component of the attack complex is thought to be amoebapore, an ion-channel forming protein that incorporates spontaneously into target cells leading to their depolarization by creating a pathway for ions to flow down their concentration gradient. The known properties of amoebapore are described. The acquisition of complement resistance by the invading trophozoites is essential for their survival within the host and therefore underlies virulence. The resistance to complement killing is not a permanent property of the amoebae. It is lost during axenization and reappears on passage through the host or when the trophozoites are grown axenically in the presence of active complement.
The lesions induced in man by Entamoeba histolytica are characterized by massive tissue injury in the absence of major local signs of a host immune response. The amoeba damages surrounding cells preferentially by contact-mediated cytolysis. Recently, a presumptive aetiological factor underlying this process has been identified. It is a protein, amoebapore, capable of spontaneous incorporation into host cell membranes. Therein it induces high conductance ion-channels which rapidly collapse the cellular transmembrane potential and lead to a prelytic state. Amoebapore is present within the amoeba in a highly aggregated state in a small, dense particle. It is shed into the medium in a particulate form by a stimulus-mediated process. Release is enhanced by addition of concanavalin A, lipopolysaccharide or the calcium ionophore A23187. Surface-labelling of intact amoeba, followed by fractionation of the homogenate in self-generating Percoll gradients, identified two labelled fractions, the plasma membrane and a particulate fraction sedimenting in the region of intracellular particulate amoebapore. This latter fraction appears to be material in the process of exocytosis. A highly immunogenic surface lipid has been identified and shown to be involved in the rapid surface redistribution of immune complexes, their shedding and endocytosis. The relevance of these findings to the immunoprophylaxis of amoebiasis is discussed.
Our aim was to verify whether differentiated murine cells grafted into mouse blastocysts would follow the same fate as teratocarcinoma cells which lose their malignant character and participate in the development of the host embryo yielding chimaeric animals. Two combinations of host embryo-cancer cells were used. Animals were examined after birth or by pregnancy interruption at various stages of development. More than 350 animals obtained from cancer-cells-injected embryos failed to show direct evidence of loss of malignancy.
Procedures are described for the synthesis of 500 A-diameter polymer microspheres containing a novel fluorescent cross-linking agent. These microspheres have very high fluorophore concentration without quenching of the fluorescence and show very low nonspecific interaction with cells. When monoclonal anti-Thy-1.2 is attached to the fluorescent microspheres, specific binding results in 10(4) spheres being attached per thymocyte while non-specific binding is less than 1%. Similar values are obtained for an indirect staining procedure. The high non-specific binding of cationic avidin to negative cell surfaces is shown to be decreased to negligible levels by acetylation of the amine groups of the protein without decreasing its high-affinity binding to biotin. The use of acetyl-avidin (pI = 6.7) directly, or when attached to fluorescent microspheres, resulted in a highly selective detection of biotinyl groups on the erythrocyte or lymphocyte cell surface. Attachment of biotinyl groups to the hinge carbohydrates of antibodies did not affect their specificity. It allowed their detection by means of microspheres-acetyl-avidin conjugates.
Hexasaminidase P, the main isozyme of hexosaminidase in pregnancy serum, was isolated and purified 600--700-fold by a two-step purification procedure--affinity chromatography on Sepharose-bound epsilon-aminocaproyl-N-acetylglucosylamine, followed by ion-exchange chromatography on DEAE-cellulose. The purified enzyme was subjected to biochemical and immunochemical analysis. Its catalytic property, namely, kinetic behavior, is similar to that of the major isozymes of hexosaminidase, A and B. However, it differs from these isozymes in its electrophoretic mobility and in its apparent molecular weight which is around 150 000 compared with 100 000 of the A and B isozymes. Immunochemical analysis indicates that the P isozymes is antigenically cross-reactive with both A and B isozymes, but it does not contain the A-specific antigenic determinants, and exhibits identical antigenic specificity to hexasaminidase B. Two possible structures are suggested that are compatible with the experimental data: (a) a hexosaminidase B like structure with higher extent of glycosylation; (b) a hexameter of beta chain, possibly arranged as three beta2 subunits.
DMSO resistant clones have been isolated from the inducible Friend leukemia cell line 5-86 both from unmutagenized cultures and following EMS mutagenesis. All the clones can grow in the presence of 1.8% DMSO and are non-inducible or poorly inducible for hemoglobin synthesis by DMSO as well as by other known inducers of Friend leukemia (FL) cells differentiation like hemin, hypoxanthine, hexamethylene bisacetamide. The clones are also defective for the expression of other properties of differentiating Friend cells like agglutinability by plant lectins and expression of the surface protein glycophorin. Some of the clones show an impaired ability to form tumors in vivo. These resistant clones might be useful for a genetic analysis of the differentiation process of Friend leukemia cells.
Frequencies of Mu-1-induced mutants of Escherichia coli have been compared under two different experimental conditions: cells in exponential growth and the same cells treated with nalidixic acid. The average of values obtained from the nalidixic acid-treated culture is 3 times higher than that obtained from the control. Individual ratios of the frequency of mutants in the two cultures yield decreasing values from 6 to 1, starting from the point of origin of DNA replication to the termini of DNA replication. These results are compatible with the idea that Mu-1 integrates at the replication fork.
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A peptide corresponding to the 11 amino acid residues of the NH2-terminal portion in the sequence of carcinoembryonic antigen(CNTHETIC CEA(1-11) peptide was attached by means of a water-soluble carbodiimide reagent to multichain poly(DL-alanine( as well as to bovine serum albumin. Both macromolecular conjugates provoked in rabbit anti-CEA(1-11) peptide antibodies. The specificity of this immunological system and the crossreactivity between the peptide and intact CEA were investigated by two methods--passive hemagglutination and modified bacteriophage inactivation. Hemmagglutination experiments showed that not only anti-CEA(1-11) sera, but also anti-CEA sera, agglutinated CEA(1-11)-coated sheep erythrocytes, and both these reactions were inhibited with CEA(1-11) peptide. In experiments with the chemically modified bacteriophage technique CEA(1-11)-coated phase was efficiently inactivated with antisera against the CEA(1-11) conjugates, and the inactivation reaction could be totally inhibited with the free peptide. The semipure CEA, but not the pure protein, could also inhibit the phage inactivation, even though less efficiently. On the basis of the above results, sera of some cancer patients were tested for their capacity to inhibit the inactivation of CEA(1-11)-coated phage by means of anti-CEA(1-11) antiserum. The results indicate that sera from a large proportion of patients with adenocarcinomas of the digestive tract, pancreas, and breast are capable of inhibiting the above inactivation, whereas most normal sera do not inhibit.
The lysogenization and induction properties of phages lambdasusN7CI857Ai7 and lambdasusN53cro27 are described. Both phages, at 32 degrees kill little, but show only a moderate frequency of lysogenization whether an amber suppressor is present or absent in the host bacterium. In the latter case, lysogens for lambdasusN7CI857Ai7 or lambdasusN53CI857cro27 can exist in two different regulatory states, here called P r- and Pr+. The Pr+ phase is characterized by phage release and cell death at 40 degrees; conversely, cells in the Pr- phase are similarly killed but release no or very little phage. Pr- is the phase usually obtained at lysogenization. Each phase may be transmitted at 32 degrees for an unlimited number of generations, however, shifts to the opposite phase take place from time to time with a low probability. Two previously described antirepressor defective mutants. Ai7 and cro27, were found to suppress specifically the growth defect caused by an amber mutation in gene N. This suppression is observed in non-suppressing hosts at 40 or 42 degrees. Apparent revertants of N- mutants were shown to be often (80%) caused by a second mutation, in the Ai gene (also called tof, cro and fed). All the revertants so far examined appeared to be recessive. Lambda phages bearing a double amber mutation in gene N did not acquire full N independence by the acquisiton of an Ai mutation; this could be achieved, however, in the presence of a CII mutation. The above findings are discussed in terms of a direct interaction between the N, Ai and CII products.
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