The Presence and Role of Transmembrane Transforming Growth Factor-α in Cultures of Rat Liver Epithelial Cells

We assessed the presence and the role of membrane TGF-α in two rat liver epithelial cell lines, either parental or transfected with c-fos proto-oncogene. c-fos overexpressing cells had more TGF-α-like activity in their membranes. When TGF-α was removed by elastase or neutralized, the growth rates of both cell lines were markedly reduced, but to a higher extent for parental cells. If membrane TGF-α seemed to play a key contribution in normal cell growth, both cell lines were unable to react to the addition of soluble TGF-α, showing that these two forms of growth factors are not equivalent.     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

Mapping of a Gene for Long QT Syndrome to Chromosome 4q25-27

Long QT syndrome (LQTS) is a heterogeneous inherited disorder causing syncope and sudden death from ventricular arrhythmias. A first locus for this disorder was mapped to chromosome 11p15.5. However, locus heterogeneity has been demonstrated in several families, and two other loci have recently been located on chromosomes 7q35-36 and 3p21-24. We used linkage analysis to map the locus in a 65-member family in which LQTS was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Linkage to chromosome 11p15.5, 7q35-36, or 3p21-24 was excluded. Positive linkage was obtained for markers located on chromosome 4q25-27. A maximal LOD score of 7.05 was found for marker D4S402. The identification of a fourth locus for LQTS confirms its genetic heterogeneity. Locus 4q25-27 is associated with a peculiar phenotype within the LQTS entity.     

Sperm nuclei analysis of a Robertsonian t(14q21q) carrier,by FISH,using three plasmids and two YAC probes

The meiotic segregation of chromosomes 14 and 21 was analysed in 1116 spermatozoa from an oligoasthenospermic carrier of a Robsertsonian translocation t(14q21q), and in 16 392 spermatozoa from a control donor, using two-colour fluorescence in situ hybridisation (FISH). Two YAC probes (cloned in yeast artificial chromosomes) specific for regions on the long arms of these chromosomes were co-hybridised. Of the spermatozoa, 12% were unbalanced, resulting from adjacent segregations. Chromosomes X, Y and 1 were also simultaneously detected in 1335 spermatozoa from the same carrier. Whereas gonosomal disomy rates were not significantly different from those of the control donors, disomy 1 were slightly but significantly increased to 0.7%. The diploidy rate was also slightly increased to approximately 1% in the translocation carrier.     

Effects of lethal irradiation and cyclosporin A treatment on the growth and tumoricidal activity of a T cell clone potentially useful in cancer therapy

The TALL-104 cell line, originally derived from a patient with T cell leukemia, can be maintained indefinitely in culture in the presence of interleukin-2 (IL-2) and is endowed with a highly potent major-histocompatibilitycomplex (MHC)-non-restricted tumoricidal activity both in vitro and in animal models. The present study analyzes in detail the short- and long-term effects of irradiation and cyclosporin A (CsA) treatment on the growth and tumoricidal function of this T cell clone as compared to polyclonal lymphokine-activated killer (LAK) cell preparations from healthy donors. DNA and RNA syntheses by both TALL-104 and LAK cells were irreversibly arrested a few hours after irradiation with 40 Gy. However, 4-h⁵¹Cr-release assays, performed on different days (day 1 to day 7) after irradiation, showed that the cytotoxic efficiency of TALL-104 cells against hematopoietic and solid tumor targets was only modestly reduced, whereas that of LAK cells was severely inhibited. Moreover, the cytotoxic responses to recombinant human IL-2 and IL-12, measured 18 h after irradiation and cytokine addition, were normal in the case of TALL-104 cells but were abolished in the case of LAK cells. Co-culture of IL-2-or IL-12-preactivated TALL-104 cells with a tumor target for 5 days in the absence of cytokines resulted in a lower efficiency of lysis, as compared to the non-irradiated effectors, especially if the initial stimulus was IL-12. These findings suggest the requirement of multiple cytokine stimulation for optimal expression of tumoricidal activity by lethally irradiated TALL-104 cells. CsA, while abrogating TALL-104 cell proliferation at the low dose of 0.5 g/ml, inhibited their cytotoxic function marginally only at high doses (100 g/ml). By contrast, CsA reduced dose-dependently the cytotoxicity of LAK cells starting at very low doses (0.5 g/ml). CsA did not impair the ability of TALL-104 and LAK cells to produce interferon (IFN), tumor necrosis factor (TNF) , and granulocyte/macrophage-colony-stimulatory factor (GM-CSF) in response to IL-2, IL-12, or tumor targets. Irradiation reduced drastically IFN production by LAK, but not TALL-104 cells; release of TNF and GM-CSF by either type of effector was inhibited by 10%–50%, depending on the stimulus. The high resistance of the TALL-104 cells' tumoricidal function to irradiation and immunosuppressive drugs renders this immortal T cell clone a potentially safe and effective reagent for new adoptive-transfer approaches to cancer in MHC-incompatible recipients.     

Saturation of penicillin-binding protein 1 by β-lactam antibiotics in growing cells of Bacillus licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β-lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.     

High frequency of-β-hexosaminidase deficiency in lymphoblastoid cell lines

The activity of seven lysosomal enzymes was determined in 25 lymphoblastoid cell lines. These lines included normal controls transformed with Epstein-Barr virus, Burkitt's lymphomas and other lymphomas with or without EBV genome.Four lines were deficient in total β-hexosaminidase activity. The deficiency was as severe as that of the variant O (Sandhoff's disease) of clinical β-hexosaminidase deficiency. The electrophoretic pattern was also similar to that observed in Sandhoff's disease.The possible mechanisms explaining the high frequency of β-hexosaminidase deficiency in lymphoblastoid cell lines are discussed.     

Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Manduca sexta

A yellow-colored protein (YCP) was isolated from the hemolymph (i.e. blood) of fifth instar wandering stage larvae of Manduca sexta. The molecular mass of YCP was 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP revealed maxima at 278 and 405 nm. Chromophore was released from YCP through denaturation of the protein with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of an ommochrome: ommatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The amino acid composition and the N-terminal sequence of YCP were determined. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that Man and GlcNAc were present in a 3:1 ratio. Circular dichroism indicated that YCP consisted of 68% beta-pleated sheet with no alpha-helices being detected. An in vitro incubation of larval fat body in the presence of [35S]methionine indicated that this organ was the site of synthesis. Ommochromes arise in insects as end products of the metabolism of tryptophan. It is well-documented that ommochromes occur in both the tissues and the excreta of insects. We propose that in M. sexta, one such tryptophan metabolite is found in the hemolymph associated with a specific protein.     

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

Summary To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.