Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes

The effect of phorbol esters on the proliferation and survival of interleukin-2(IL-2)-dependent cells was studied using an IL-2-dependent T cell line (CTLL-2) and blasts of BALB/c mouse spleen cells stimulated with Concanavalin A. Addition of phorbol 12,13-dibutyrate (PDBu) to CTLL-2 or ConA blasts induces a mitogenic response which is 25-40% of that elicited by IL-2. Interleukin 2 deprivation leads to a marked decline in the number of viable cells (50% of CTLL-2 cells have died after 8-10 hours incubation in IL-2-free medium). The mechanism of cell death seems to correspond to the suicide process known as apoptosis since an early degradation of DNA into oligonucleosome-size fragments could be observed after removal of the growth factor. When present, PDBu inhibits both the activation of the endonuclease and the development of the cell death process in CTLL-2 cells and ConA-blasts deprived of IL-2. Taken together, our results suggest that the tumor promoters phorbol esters inactivate in T cells the mechanism of cell elimination triggered by IL-2 deprivation and may help to explain why transformation of T cells decreases or even abolishes their requirements of IL-2 for survival and growth.     

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.     

Identification of positive and negative regulatory regions controlling expression of the cartilage matrix protein gene.

A complex pattern of regulation of the cartilage matrix protein gene was revealed by transient expression experiments. A minimal promoter from positions -15 to +64 functioned in chondrocytes and fibroblasts. An enhancer located in the first intron exerted chondrocyte-specific stimulation on the minimal promoter activity. The same fragment, however, had a negative effect in fibroblasts. Between -334 and -15, a silencer was found which inhibited the gene expression driven from its homologous as well as heterologous promoters both in chondrocytes and fibroblasts. Additional positive and negative control regions were mapped further upstream of the promoter.     

Influence of the solvent on the conformational-dependent properties of random coil polypeptides. II. Mean square optical anisotropies and Kerr constants

Mean square optical anisotropies and molar Kerr constants were calculated for homopolypeptides of the 20 natural amino acids and of several enzymes and proteins in the random-coil state. The effect of hydration was taken into account in constructing the molecular potential that gives the conformational energies as a function of the rotational angles phi and psi of the backbone and chi(1) of the side chain. The Rotational Isomeric State model was used in calculated energies, the Valence Optical Scheme and the matrix calculus technique of Flory being employed in the evaluation of the optical properties. The results are compared with calculations for the same substances that were performed without taking into account the solvent, as well as with other similar studies. The Kerr constant is confirmed as being one of the most sensitive properties of a given polypeptide to the residue class and to the sequence of those residues.     

The fluorescence induction kinetics as a non-destructive tool for investigating spruce treated with ozone

Summary The scattering coefficient of yellow spruce needles exceeds that of green needles by a factor of 2, whereas the fluorescence efficiency is approximately equal for both needle colours. As shown by the angular distribution the fluorescence light is diffusely emitted. However, the scattered light consists of a diffuse and a reflecting portion below 20° with a ratio of the intensities of 1 : 2 at perpendicular observation (0°). Control measurements show that in the rejection region the effective transmission of cut-off-filters commonly used to separate fluorescence light and excitation light exceeds the value calculated from the filter specifications by a factor of 100. Therefore, the portion of the scattered light in the measuring signal must be controlled if the fluorescence induction kinetics is measured from specimen of different colour. A device for the determination of the fluorescence induction kinetics is described which employs a He-Ne laser, a mechanically working shutter with an opening time of 4 ms for the excitation, and a computer for data storage and device control. Two filters select the fluorescence components at 685 nm and 730 nm and they reduce the portion of the scattered light in the measuring signal to 0.18% and 0.55%, respectively. In order to consider the temporal development of the fluorescence kinetics the sampling rate is reduced from 2 kHz to 1 Hz. From the data stored in the computer maximum valueF _P, and steady-state-valueF _S are determined for both fluorescence components. Measurements on 4-year-old spruce exposed to ozone-concentrations of 0, 300 ppb, 600 ppb, and 1000 ppb were repeated every week. With increasing concentration and duration of treatmentR _fd =(F _P-F_s)/F _S was decreased for both fluorescence components. With the highest ozone concentration a reduction ofR _fd of 23% and 24%, respectively, was obtained for the two fluorescence components after three weeks.     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.     

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.     

In vitro depolymerization dynamics of brain endogenous microtubules

A subcellular fraction containing fragments of endogenous microtubules stabilized in 50% glycerol was separated by diferential centrifugation of rat brain homogenates. The pellets were suspended in glycerol-deficient media, and microtubule depolymerization was monitored by measuring the decrease of sedimentable tubulin. Concomitantly, the number and size of microtubules in the suspensions were followed via electron microscopy. Depolymerization was accompanied by a proportional decrease in the number of microtubules, whereas the average size did not change significantly. After approximately 20 min, a subpopulation of microtubules became stable and did not suffer further depolymerization. These results indicate that upon dilution some microtubules completely depolymerize, whereas others remain stable in the glycerol-deficient medium. The degree of depolymerization depended on both the volume of the resuspension media and on the final glycerol concentration. The results suggest that the depolymerization of the remaining microtubules is prevented by stabilizing factors released from depolymerizing microtubules. Tubulin dimers are not one of these factors, since depolymerization was not altered by the addition of colchicine or by changing the concentration of free tubulin in the medium.