Antidepressants attenuate the dexamethasone-induced decrease in viability and proliferation of human neuroblastoma SH-SY5Y cells: a involvement of extracellular regulated kinase (ERK1/2).

Excessive glucocorticoid levels in depressed patients have been associated with atrophic changes in some brain regions, but only few studies suggest that some antidepressants can interfere with deleterious effect of glucocorticoids on neuronal cells. The aim of the present study was to examine the effect of dexamethasone (DEX), a synthetic glucocorticoid and some antidepressants from different chemical groups (imipramine, desipramine, amitriptyline, citalopram, fluoxetine, reboxetine and tianeptine) on SH-SY5Y cells cultured in the medium containing steroid-free serum. DEX in concentrations from 1 to 100 µM did not change LDH release but exposure to 10 µM and 100 µM DEX for 24, 48 and 72 h caused a significant reduction in cell viability and proliferation as confirmed by MTT reduction and BrdU ELISA assays, respectively. Twenty four-hour incubation of cells with antidepressants (0.05-10 µM) and DEX (10 µM) showed that imipramine, amitriptyline, desipramine, citalopram and fluoxetine at concentrations from 0.1 up to 1 µM, reboxetine (0.1 µM) and tianeptine (0.05 µM) prevented the DEX-induced decreases in cell viability and proliferation rate. The protective effects of antidepressants were ameliorated by inhibitors of MAPK/ERK1/2, but not PI3-K/Akt pathway as shown for imipramine, fluoxetine and reboxetine. Moreover, Western blot analysis showed the decrease in the activated form of ERK1/2 (p-ERK) after DEX treatment and this effect was inhibited by imipramine. Thus, the reduction in SH-SY5Y cell viability caused by DEX appears to be related to its antiproliferative activity and some antidepressant drugs in low concentrations attenuate this effect by mechanism which involves the activation of MAPK/ERK1/2 pathway.

Molecular subdivision of the cortex of dividing Tetrahymena is coupled with the formation of the fission zone.

In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized ciliates is preceded by a reorganization of the cortex into dual metameric patterns for prospective daughter cells and then separated by a transverse fission line. This study concerns relations between the generation of cortical metamery and the formation of the fission line in an amicronuclear (i.e., without mitotic spindle) ciliate, Tetrahymena pyriformis. The fission line appears in the division of T. pyriformis as a transverse line formed by equatorial gaps in the meridional ciliary rows, with the second oral structure (OA2) formed posterior to it. It was found that the metamery of cortical morphogenesis is expressed by the appearance of increased MPM2 antibody binding in dividing cells in an apical area and posterior to the fission line gaps, including patterned changes of this binding in both oral apparatuses (OA1 and OA2), and by a reciprocal decrease of binding of an anti-epiplasm antibody. These tested antigens are localized to different cortical structures, but in predividing cells both uniformly show formation of the fission line contrast of labeling. A serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was applied to dividing T. pyriformis at specific stages: (1) if 6-DMAP was added to early dividing cells, it prevented cells from initiating cytokinesis. (2) If 6-DMAP was added to cells at stages close to the physiological transition point of cell division, it yielded either (i) a partial formation of the fission line on the ventral side, combined with modified growth of undivided cortex adjacent to the fission line, with abnormal cytokinesis, or (ii) variable anterior displacement of the complete fission line, which contracted slowly but uniformly. (3) If 6-DMAP was applied during cytokinesis, it did not delay cell division, but daughter cells become abnormal and underwent an incomplete oral reorganization. These results suggest that the generation of metamerism in the cortex of T. pyriformis involves differentiation of the asymmetric fission zone. At least four stage-dependent 6-DMAP-sensitive effects jointly control the progress of cell division and the mutual spatial relations between the generation of metamery and the appearance, completeness, and position of the fission zone in the cortex of polarized T. pyriformis.

Induction of blocks in nuclear divisions and overcondensation of meiotic chromosomes with cycloheximide during conjugation of Tetrahymena thermophila.

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I-III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).

Relationship between spatial pattern of basal bodies and membrane skeleton (epiplasm) during the cell cycle of Tetrahymena: cdaA mutant and anti-membrane skeleton immunostaining.

Microtubular basal bodies and epiplasm (membrane skeleton) are the main components of the cortical skeleton of Tetrahymena. The aim of this report was to study functional interactions of basal bodies and epiplasm during the cell cycle. The cortex of Tetrahymena cells was stained with anti-epiplasm antibody. This staining produced a bright epiplasmic layer with a dark pattern of unstained microtubular structures. The fluorescence of the anti-epiplasm antibody disappeared at sites of newly formed microtubular structures, so the new basal body domains and epiplasmic layer could be followed throughout the cell cycle. Different patterns of deployment of new basal bodies were observed in early and advanced dividers. In advanced dividers the fluorescence of the epiplasmic layer diminished locally within the forming fission line where the polymerization of new basal bodies largely extincted. In wild type Tetrahymena, the completion of the micronuclear metaphase/anaphase transition was associated with a transition from the pattern of new basal body deployment and epiplasm staining of the early divider to the pattern of the advanced dividers. The signal for the fission line formation in Tetrahymena (absent in cdaA1 Tetrahymena mutationally arrested in cytokinesis) brings about 1) transition of patterns of deployment of basal bodies and epiplasmic layer on both sides of the fission line; and 2) coordination of cortical divisional morphogenesis with the micronuclear mitotic cycle.

The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila.

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35 degrees C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity.

Macronuclear differentiation in conjugating pairs of Tetrahymena treated with the antitubulin drug nocodazole.

During Tetrahymena conjugation gamic nuclei (pronuclei) are produced, reciprocally exchanged, and fused in each mate. The synkaryon divides twice; the two anterior nuclei develop into new macronuclei while the two posterior nuclei become micronuclei. The postzygotic divisions were blocked with the antitubulin drug nocodazole (ND). Then pronuclei (gamic nuclei) developed directly into macronuclear anlagen (primordial macronuclei), inducing amicronucleate cells with two anlagen, or, rarely, cells with one anlagen and one micronucleus. ND had a similar effect on cells that passed the first postzygotic division inducing amicronucleate cells with two anlagen, while cells treated with ND at the synkarya stage produced only one large anlage. Different intracytoplasmic positioning of the nuclei treated with ND (pronuclei, synkarya and two products of the first division) shows that most of cell cytoplasm is competent for inducing macronuclear development. Only posteriorly positioned nuclei--products of the second postzygotic division--remain micronuclei. The total cell DNA content, measured cytophotometrically in control and in ND-induced amicronucleate conjugant cells with one and two anlagen, was similar in all three samples at 12 h of conjugation. Eventually, at 24 h this content was about 2 pg (8 C) per anlagen both in nonrefed control and in amicronucleate exconjugants. Therefore "large" nuclei developing in the presence of ND were true macronuclear anlagen.

The formation of basal body domains in the membrane skeleton of Tetrahymena.

Differentiated regions within the membrane skeleton are described around basal bodies in the ciliary rows of Tetrahymena. These domains, approximately 1 micron in diameter, are characterized by a relatively dense ultrastructure, the presence of a family of proteins called K antigens (Mr 39-44 x 10(3)) that are recognized by mAb 424A8, and the apparent exclusion of major membrane skeleton proteins that are present in most other regions of the cell (Mr 135, 125 x 10(3]. Mature basal body domains are asymmetric, reflecting the polarity of the cell as a whole. A similar differentiation of the membrane skeleton occurs in the oral apparatus, except here the K antigens surround four clusters of basal bodies (from which this cell takes its name) rather than the individual basal bodies. The development of new basal body domains in the cell cycle is described, with similarities and differences noted between somatic and oral regions of the cell. It is concluded that the capacity of this cell for precise topographic regulation of molecular events in the membrane skeleton makes it a useful model for the study of cortical differentiation in cells generally.

Alterations of blood platelet function induced by neutral proteases from human leukocytes.

Washed human platelets are damaged by two neutral proteases from human leukocytes (elastase-like protease, ELP and chymotrypsin-like protease, CLP). The damage is manifested as inhibited aggregation by ristocetin and collagen, and enhanced aggregation by ADP in the presence of fibrinogen. Similarly to alpha-chymotrypsin (alpha-CT), CLP also increases binding of 125I-fibrinogen to platelets and renders platelets aggregable by human fibrinogen. ELP is less effective in this respect possibly due to damage to platelet receptors for fibrinogen. In the plasma medium platelets are not sensitive to leukocytic proteases added at concentrations that provoke some prolongation of the time of plasma clotting by thrombin.

Effects of neutral proteases from human leukocytes on plasma fibronectin.

Human plasma fibronectin (FN) is not susceptible to collagenase and gelatinase from human blood leukocytes. Leukocytic elastolytic protease (ELP) and cathepsin G (chymotrypsin-like protease, CLP) degrade FN to similar fragments. Among products of proteolysis by ELP and CLP fragments have been identified which bind to gelatin-fragment 40 kd, to fibrin-fragments 55 kd and 30 kd, and to heparin-fragment 30 kd.

Effect of neutral proteases from blood leukocytes on human platelets.

Two highly purified neutral proteases from human leukocytes i.e. elastase-like protease (ELP) and chymotrypsin-like protease (CLP) do not destroy human platelets since no difference was found in 51Cr liberation from control and enzyme-treated platelets. As with pancreatic chymotrypsin (alpha-CT) ELP does not induce the release of 3H-serotonin while CLP provokes 3H-serotonin secretion, in an enzyme concentration and time dependent fashion. The rate and degree of 3H-serotonin release by CLP is similar to that produced by thrombin. Incubation of platelets at 37 degrees C for 30 min with alpha-CT or ELP renders them resistant to thrombin-releasing activity. Thrombin did not liberate any additional label from platelets which lost over 60% of serotonin during the preceding incubation with CLP. alpha-CT and ELP do not aggregate platelets either in the presence or absence of apyrase. CLP does aggregate platelets suspended in Tyrode buffer without apyrase but not in the presence of apyrase (100 mg/l). The action of alpha-CT, ELP and CLP on washed platelets induces a progressive prolongation of lag phase and a decrease in changes of light transmission during aggregation by thrombin. Similarly to alpha-CT-treated platelets, those subjected to CLP action aggregate in the presence of human fibrinogen. It is concluded that: (1) neutral proteases possibly contribute to development of defects in platelet function in pathological states associated with liberation of leukocyte content into the circulation, (2) CLP similarly to alpha-CT, exposes fibrinogen receptors but in contrast to alpha-CT, CLP aggregates platelets and stimulates serotonin secretion.

Pattern determination and pattern regulation in Paramecium tetraurelia.

Pattern regulation was investigated in the progeny of laterally fused cells of Paramecium tetraurelia. The immediate progeny of such fused cells (doublets) reveal two sets of cortical organelles arranged roughly symmetrically. Doublets tend to transform gradually into cells with only one set of organelles (singlets). At least two different and mutually exclusive pathways of doublet-to-singlet transformation are reported. In intermediate stages of regulation the cortical areas bearing different cortical landmarks may be brought into an abnormal neighbourhood. Differentiated cortical bands of cortex, bearing organellar landmarks, are faithfully propagated even if they are improperly and asymmetrically located on the cell. The confrontation of such cortical bands may lead to the transient appearance of additional duplicated organelles. It is suggested that pattern regulation in Paramecium during doublet-to-singlet transformation results from at least three factors: the regression of some part of the cortical areas, the interaction of the juxtaposed parts remaining and the slow regulatory shift of positions of the cortical structures.

Polymorphism and specificity of positioning of contractile vacuole pores in a ciliate, Chilodonella steini.

The unicellular ciliate Chilodonella steini has a well-defined flat and ciliated ventral field. During divisional morphogenesis two sets of new contractile vacuole pores (CVPs) are formed on this field. During final pattern formation some of these CVP primordia and the old parental set of CVPs are completely resorbed. Primary patterns of distribution of the CVP primordia and final pattern of distribution of the matured CVPs manifest an intraclonal polymorphism. From analysis of this polymorphism some features of mechanism(s) of CVP pattern determination are deduced: 1. There is a strict, short-distance negative control of appearance of CVP primordia at sites of oral morphogenesis and around the ventral field. 2. Certain indeterminacy of large-scale patterning of CVP primordia is observed over the area competent to yield CVP formation. However, within this area three longitudinal sectors with a high probability of occurrence of CVP primordia are alternated with sectors nearly deprived of their occurrence. 3. Positive control of probability of occurrence and of specificity of location is found for certain CVP primordia. An interaction of mechanism of positioning on cellular longitudes and latitudes is proposed to account for these facts. 4. The resorption of supernumerary CVP primordia does not alter the character of the global map of distribution of CVP primordia achieved during primary pattern formation. The primordia located at some latitudes persist, whereas others are resorbed at random. It is suggested that all CVP primordia which do not mature at the time of stabilization of divisional morphogenesis are resorbed. Thus the global map of CVPs distribution would result from the sum of the individual determinations of the fates of each CVP primordium, superimposed on an initial spatially non-uniform distribution of CVP primordia.

Effects of neutral proteases from human leukocytes on structure and biological properties of human factor VIII.

Human factor VIII was purified from cryoprecipitate and incubated for up to 24 hours with four neutral proteases of human blood leukocytes, namely, with elastase-like protease (ELP), chymotrypsin-like protease (CLP), collagenase and gelatinase. Electrophoretic patterns showed a reproducible sequence of degradation of factor VIII and of its 230,000 molecular weight subunit by ELP and CLP. Intermediate products were similar but those resulting from exhaustive proteolysis by ELP and CLP differed distinctly from each other. Procoagulant activity of factor VIII was rapidly and completely destroyed by ELP and CLP before visible electrophoretic changes would be detected. No increase in this activity was observed prior to its destruction. Von Willebrand factor (ristocetin cofactor) activity was considerably more resistant to ELP and CLP and declined in rough relation to degradation of highly aggregated forms of factor VIII. ELP and CLP produced a pronounced progressive increase in the Laurell reaction antigen. Normal human plasma showed a high potency to inhibit ELP and CLP. Large doses of these enzymes (300 microgram per ml) produced in the plasma medium only a moderate fall in factor VIII procoagulant activity. Collagenase and gelatinase did neither degrade factor VIII nor change its biological properties.

Physiological dissection of various effects of ruthenium red dye on Paramecium cells.

Polycationic dye ruthenium red, but not alcian blue, if externally applied to Paramecium cells quickly inhibits their phagocytosis. Ruthenium red combined with the cell surface diminishes frequency and duration of ciliary reversals and gradually inactivates the Ca++ gating mechanism. This effect persists for 1-3 after ruthenium red removal from the culture medium.

Effects of actinomycin D on generation time and morphogenesis in Paramecium.

The sensitivity of Paramecium tetraurelia (=P. aurelia syngen 4) cells to pulse treatments with various doses of Actinomycin D (AMD) was estimated by comparing the generation times of treated and untreated sister cells. It was found that the delay of division in treated cells depended on the concentration of AMD, on their "age" at the time of the pulse treatment, and on their individual sensitivity. Sensitivity of Paramecium to AMD changes during the cell cycle in a predictable way. About 3 1/2 hr before the normally expected cell fission (total generation time approximately 5 1/2 hr) there is a decrease of sensitivity. Thereafter, the cell enters a new stage with a progressive increase of sensitivity. This 2nd phase ends at the "transition point" (approximately 2 hr before cell division), when sensitivity drops abruptly. The division process itself may be altered and slowed down by high concentrations of AMD, even if the drug is applied after the transition point, but this process can never be completely annulled. The impairment of the division mechanism may lead to morphologic anomalies in the offspring. Resorption of oral anlagen in P. tetraurelia probably never occurs during the cell cycle after AMD treatment. The reason for individual variability of the cells, mechanisms controlling development, and the question of an obligate sequence of gene action in each cell cycle are discussed.