Lysosomal hydrolase activity in chorionic villi and embryonic cells in culture

Summary Fourteen lysosomal enzymes were compared in 20 cultured cell lines from chorionic biopsy and corresponding embryonic tissue after voluntary abortions. Enzymatic expression appears to be similar in cultured cells from both sources with some slightly higher levels for chorionic villi. We stress the importance of culturing chorionic villi especially in the case of enzymes (-L-iduronidase) or diseases (I cell disease) whose expression is unusual in fresh trophoblast tissue.     

Mechanism of α Factor Biosynthesis in Saccharomyces cerevisiae

The biosynthesis of alpha factor, a mating-type-specific regulatory oligopeptide which is secreted by Saccharomyces cerevisiae cells of alpha mating type, was studied. In batch cultures only small amounts of the peptide were synthesized during the exponential growth phase. During the stationary phase, alpha factor was produced at a constant rate and accumulated in the culture medium. Inhibition of translation in wild-type cells by cycloheximide, or in mutant strains under conditions which blocked protein or ribonucleic acid (RNA) synthesis completely inhibited the production of alpha factor. These results indicate that the factor is produced by ribosomal translation of a specific messenger RNA and not by an extraribosomal mechanism of peptide synthesis.     

Genetic structure of natural populations ofDryas iulia (Lepidoptera: Nymphalidae) revealed by enzyme polymorphism and mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP)

Dryas iulia appears to have undergone a mode of evolution different from that of other members of its subfamily (Heliconiinae). While other species constitute highly subdivided and inbred populations, those ofD. iulia are thought to be large and uniform. Analyzing six samples from Southern Brazil (state of Rio Grande do Sul) in relation to three enzyme systems (EST, LAP, and PGM) and their mtDNA RFLP patterns, we found that they are very similar at the molecular level. TheF statistics for enzyme polymorphism data revealed that inbreeding makes a great contribution to the population homozygosity, sinceF _IS equals 0.1322 andF _ST equals 0.0023. Since the chi-square test showed thatF _ST is not significant, we conclude that all localities belong to the same population. The mtDNA differentiation was about 12 times greater than for nuclear genes;F _ST was equivalent to 0.0265. We suggest that this difference is due to a higher dispersal of males, in relation to females.     

Female reproductive fluid concentrations affect sperm performance of alternative male phenotypes in an external fertilizer

There is growing evidence that the female reproductive fluid (FRF) plays an important role in cryptic female choice through its differential effect on the performance of sperm from different males. In a natural spawning event, the male(s) may release ejaculate closer or further away from the spawning female. If the relative spatial proximity of competing males reflects the female pre-mating preference towards those males, then favoured males will encounter higher concentrations of FRF than unpreferred males. Despite this being a common situation in many external fertilizers, whether different concentrations of FRF can differentially influence the sperm performance of distinct male phenotypes (favoured and unfavoured by the female) remains to be elucidated. Here, we tested this hypothesis using the grass goby (Zosterisessor ophiocephalus), a fish with distinct territorial-sneaker reproductive tactics and female pre-mating preference towards territorial males, that consequently mate in an advantaged position and whose sperm experience higher concentrations of FRF. Our findings revealed a differential concentration-dependent effect of FRF over sneaker and territorial sperm motility only at low concentrations (i.e. at the distance where sneakers typically ejaculate), with increasing FRF concentrations (i.e. close to the eggs) similarly boosting the sperm performance of both sneaker and territorial males. The ability to release sperm close to the eggs is a prerogative of territorials, but FRF can likewise advantage the sperm of those sneakers that are able to get closer, allowing flexibility in the direction of female post-mating choice.     

The intrinsic electrophysiological characteristics of fly lobula plate tangential cells: I. Passive membrane properties

The passive membrane properties of the tangential cells in the fly lobula plate (CH, HS, and VS cells, Fig. 1) were determined by combining compartmental modeling and current injection experiments. As a prerequisite, we built a digital base of the cells by 3D-reconstructing individual tangential cells from cobalt-stained material including both CH cells (VCH and DCH cells), all three HS cells (HSN, HSE, and HSS cells) and most members of the VS cell family (Figs. 2, 3). In a first series of experiments, hyperpolarizing and depolarizing currents were injected to determine steady-state I-V curves (Fig. 4). At potentials more negative than resting, a linear relationship holds, whereas at potentials more positive than resting, an outward rectification is observed. Therefore, in all subsequent experiments, when a sinusoidal current of variable frequency was injected, a negative DC current was superimposed to keep the neurons in a hyperpolarized state. The resulting amplitude and phase spectra revealed an average steady-state input resistance of 4 to 5 M and a cut-off frequency between 40 and 80 Hz (Fig. 5). To determine the passive membrane parameters R _m (specific membrane resistance), R _i (specific internal resistivity), and C _m (specific membrane capacitance), the experiments were repeated in computer simulations on compartmental models of the cells (Fig. 6). Good fits between experimental and simulation data were obtained for the following values: R _m = 2.5 kcm², R _i = 60 cm, and C _m = 1.5 F/cm² for CH cells; R _m = 2.0 kcm², R _i = 40 cm, and C _m = 0.9 F/cm² for HS cells; R _m = 2.0 kcm², R _i = 40 cm, and C _m = 0.8 F/cm² for VS cells. An error analysis of the fitting procedure revealed an area of confidence in the R _m -R _i plane within which the R _m -R _i value pairs are still compatible with the experimental data given the statistical fluctuations inherent in the experiments (Figs. 7, 8). We also investigated whether there exist characteristic differences between different members of the same cell class and how much the exact placement of the electrode (within ±100 m along the axon) influences the result of the simulation (Fig. 9). The membrane parameters were further examined by injection of a hyperpolarizing current pulse (Fig. 10). The resulting compartmental models (Fig. 11) based on the passive membrane parameters determined in this way form the basis of forthcoming studies on dendritic integration and signal propagation in the fly tangential cells (Haag et al., 1997; Haag and Borst, 1997).     

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.     

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.