The Effect of Colchicine and Cytochalasin B on the Release of Taurine from the Chick Retina

Abstract: The effect of colchicine (0.5 mM) and of cytochalasin B (10⁻⁴ M) on the release of [³⁵S]taurine from the isolated chick retina, upon stimulation by 68.5 mM-KCl, 10⁻⁵ M-veratridine and 10 mM-glutamate, was studied. Cytochalasin and colchicine effects on taurine release were compared with those on K⁺-stimulated release of [³H]dopamine and [³H]GABA. Colchicine caused a marked decrease of the [³⁵S]taurine release evoked by the three stimulatory agents; it also decreased [³H]dopamine release without affecting that of [³H]GABA. Cytochalasin B significantly decreased the efflux of [³⁵S]taurine stimulated by glutamate or veratridine without altering that evoked by 68.5 mM-KCl. Cytochalasin practically suppressed the [³H]dopamine-stimulated release and slightly decreased that of [³H]GABA. This drug produced a high increase in the spontaneous release of labeled GABA and taurine. These results suggest that the release of taurine and GABA from the chick retina probably occurs through different mechanisms. It is suggested that taurine release may be related to a process involving contractile proteins.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁     

Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody

Abstract We have recently shown that frizzled-9 (FZD9, CD349) is expressed on the cell surface of cultured mesenchymal stromal cells (MSC) derived from the human bone marrow (BM) and chorionic placenta (PL). To study whether FZD9 is also a marker for naive mesenchymal stem cells (MSC), we analyzed the expression pattern of FZD9 on freshly isolated PL cells and determined the clonogenic potential of isolated FZD9⁺ cells using the colony-forming units-fibroblastic (CFU-F) assay. About 0.2% of isolated PL cells were positive for FZD9. Two-color analysis revealed that FZD9⁺ PL cells uniformly express CD9, CD63, and CD90, but are heterogeneous for CD10, CD13, and CD26 expression. In contrast to BM-derived MSC, PL-derived MSC expressed only low levels of CD271. Colony assays of sorted cells showed that clonogenic CFU-F reside exclusively in the FZD9⁺ but not in the FZD9⁻ fraction. Further analysis revealed that CFU-F were enriched by 60-fold in the FZD9⁺CD10⁺CD26⁺ fraction but were absent in the FZD9⁺CD10⁻CD26⁻ population. Cultured FZD9⁺ cells expressed the embryonic stem cell makers Oct-4 and nanog as well as SSEA-4 and TRA1-2-49/6E. In addition, they could be differentiated into functional adipocytes and osteoblasts. This report describes for the first time that FZD9 is a novel and specific marker for the prospective isolation of MSC from human term PL.     

MICROINJECTION OF COLCHICINE INTO SEA URCHIN EGGS

Inhibition of cleavage by colchicine was examined by microinjecting colchicine solution into one of the blastomeres of a sea urchin egg at the two-cell stage. Cleavage was inhibited if the microinjection was made before a critical point prior to the cleavage, whereas cleavage occurred in spite of the destruction of the mitotic apparatus if the microinjection was made after the critical point. The critical point was 10 min before the mid-stage of the cleavage in Clypeaster japonicus and 8 min before the mid-stage in Temnopleurus toreumaticus at 20 ± 1°C, corresponding to the beginning of anaphase. The threshold for the cleavage inhibition of colchicine was estimated to be 3 × 10⁻⁵ M to 3 × 10⁻⁶ M in final concentration in the cell.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

Permeability of the Chara cell membrane for ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol

The permeability of internodal cells of Chara australis R. Brown for polyol molecules was determined by using a turgor balance to measure the increase in the osmotic pressure of an internodal cell incubated in artificial pond water containing one of the polyol compounds tested. The permeabilities for ethylene glycol, glycerol, meso -erythritol, xylitol and mannitol were (4. 39 ± 0. 52) × 10⁻⁹, (1. 49 ± 0. 40) × 10¹⁰, (4. 92 ± 0. 27) × 10⁻¹⁰ (9. 9 ± 3. 4) × 10¹¹ and (7. 6 ± 4. 8) × 10⁻¹² m s⁻³, respectively. The permeability for glycerol was slightly smaller than that for meso -erythritol, whose molecular weight is larger than that of glycerol in this homologous series: but the reason for this is not clear.     

Binding of cytotoxin P4 from Naja nigricollis nigricollis to B16F10 melanoma and WEHI-3B leukemia cells

Abstract Cobra venoms cause irreversible destruction of cells cultured in vitro [1,2]. The venom of Naja nigricollis nigricollis possessed the most potent cytotoxic activity towards B16F10 melanoma cells among various examined venoms [2]. The main cytotoxic factor (P4) isolated from this venom showed preferential activity on tumor cell lines and caused lysis at concentrations of 10⁻⁷ M (0.8–1 μg/ml) [3]. The present study examined the binding of cytotoxin P4 to melanoma B16F10 and WEHI-3B leukemia cell lines and found that, like cytotoxicity, it depended on concentration, temperature and incubation time. Cytotoxin concentrations that elicited no apparent damage to cells during the first hour of incubation caused lysis after a longer period of incubation, suggesting that a critical number of bound molecules is required in order to cause cell death. Bivalent ions, such as Mg²⁺, Ca²⁺ or Sr²⁺, which decreased binding to the cells also inhibited cytotoxicity. Competition experiments as well as the displacement of 75% of the bound radiolabelled cytotoxin with 'cold' cytotoxin, suggest the presence of specific binding sites for the toxin in the examined tumor cells. The non-specific binding of the cytotoxin P4 to sea urchin ova and sperm cells without affecting their fertility, even at high concentrations of 10⁻⁵ M, indicates that the specific binding to cells is probably a necessary condition for cell lysis.     

Efficacy of certain disinfectants against infectious pancreatic necrosis virus

The virucidal properties of iodophor, chlorine (sodium hypochlorite), formalin, thimerosal (organic mercurial compound), malachite green, and acriflavine were tested on infectious pancreatic necrosis virus (IPNV). Iodine and chlorine showed good activity, but efficacy depended on the concentration of virus, the presence of organic matter (calf serum), and water _pH. Water hardness (0-300 mg 1⁻¹ as CaCO₃) did not affect virucidal activity. In a 5 min exposure, 4 mg 1⁻¹ available iodine inactivated 10^3.9 TCID₅₀ m1⁻¹ IPNV but 16 mg 1⁻¹ iodine were needed for inactivation of 10^6.3TCID₅₀m1⁻¹. The addition of 0-5% calf serum significantly reduced the iodine concentration and the virucidal activity. In comparison, 4 mg 1⁻¹ chlorine were needed to inactivate 10⁴⁶ TCID₅₀ m1⁻¹ IPNV in 5 min. However, the addition of 0-07 % serum greatly reduced the chlorine concentration and extended the virucidal contact time to 30 min or more. IPNV at 10^6.3 TCID₆₀ m1⁻¹ was not inactivated by exposures for 60 min to 0-2% formalin, 10 min to 0-2% thimerosal, 60 min to 5 mg 1⁻¹ malachite green, or 20 min to 500 mg 1⁻¹ acriflavine. However, acriflavine at 0-5 mg 1⁻¹ in cell culture media prevented the development of cytopathology caused by IPNV and may be useful in the treatment of the disease.     

Exoprotease activity of Leuconostoc oenos in stress conditions

Exoprotease activity during 48 h of total energy and nutrient starvation was examined in Leuconostoc oenos X₂L isolated from wine. Starved cells after 2 h of incubation at 30 °C in citrate buffer, 0.05 mmol 1⁻¹ pH 5, showed greater extracellular proteolytic activity than at the onset of starvation. In the presence of 60 mg l⁻¹ SO₂ and 8% or 12% ethanol, the proteolytic activity was higher ; 10 mmol l⁻¹ Ca²⁺ and Mg²⁺ produced an increase in protease activity during starvation. Glucose and 2-deoxyglucose (2-DOG) were found to repress synthesis by 80% and 100%, respectively. Cyclic adenosine 3'-5'-phosphate increased the exoprotease activity and reverted the repression by glucose and 2-DOG. De novo synthesis of proteins was required for the exoprotease activity by cells submitted to stress conditions. The absence of protease activity in the supernatant fluids from chloramphenicol-treated cells indicated that the activity is a result of deliberate release and not of passive cell lysis.     

Conjugal transfer at natural population densities in a microcosm simulating an estuarine environment

Abstract Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. Donor cells were added at a concentration similar to the natural abundance of bacteria in the water column (10⁶ cells ml⁻¹). Transfer was not detected in any of the test microcosms (calculated limit of detection of 10⁻⁷ and 10⁻⁴ transconjugants donor⁻¹ in water column and sediment, respectively), with the exception of transfer to an isogenic recipient (added at 10⁵ cells ml⁻¹) in sediments of controls that had been inoculated with both donors and recipients. The same plasmid was transferred with high efficiencies (10⁻¹ to 10⁻³) to a variety of recipients in filter and broth matings. These results suggest that if conjugal gene transfer occurred, it was at efficiencies that were not detectable in estuarine microcosms simulating natural population densities.     

MORPHOLOGICAL CHANGES OF CULTURED MYOCARDIAL CELLS DUE TO CHANGE IN EXTRACELLULAR CALCIUM ION CONCENTRATION

Increase in the extracellular Ca²⁺ concentration from low (≤ 10⁻⁷ M) to normal (10⁻³ M) caused morphological changes of cultured myocardial cells obtained from fetal mouse heart. The extracellular Na⁺ and K⁺ concentrations of the normal medium (10⁻³ M Ca²⁺) did not significantly affect the genesis of these morphological changes. Like Ca²⁺, Ba²⁺ and Sr²⁺, but not Mg²⁺, Co²⁺ or Ni²⁺, could induce morphological changes. Increase in the extracellular Ca²⁺ concentration from 10⁻⁸ M to 10⁻³M also caused excess uptake of ⁴⁵Ca²⁺ by cultured myocardial cells. B–16CW 1 cells, which did not show these morphological changes, did not take up excess ⁴⁵Ca²⁺ on this treatment. Treatments, such as addition of verapamil or incubation at pH 6.3, which reduced the genesis of morphological changes, reduced the rate of ⁴⁵Ca²⁺ uptake by myocardial cells. These facts show that the morphological changes of myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal are due to excess uptake of Ca²⁺ by the myocardial cells.
The morphological changes of cultured myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal were reversed on further incubation of the cells in medium with or without Ca²⁺.     

The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli

Abstract The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli . The dnaJ ⁻ cbpA ⁻ double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ ⁻ cbpA ⁻~ mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD , the gene that encodes the major σ ⁷⁰. The biological implications of this finding are examined and discussed.     

Potassium transport in wheat seedlings grown with different potassium supplies.

The K⁺(⁸⁶Rb) uptake into the roots and the translocation to the shoots of 11-day-old intact wheat seedlings ( Triticum aestivum L. cv. Martonvásári 8) were investigated using plants grown with different K⁺ supplies. The effects of environmental conditions (darkness, humidity) and of metabolic and transport inhibitors (oligomycin, disalicylidene-propanediamine, 2,4-dinitriphenol, diethylstilbestrol, colchicine) were also studied. Plants with K content of about 0.2 mmol/g dry weight in the root and 0.5 mmol/g dry weight in the shoot (low K status) showed high K⁺ uptake into the roots and high translocation rates to the shoots. Both transport processes were very low in plants with K content of more than 1.5 and 2.2 mmol/g dry weight in the root and shoot, respectively (high K status).
Darkness and a relative humidity of the air of 100% did not influence K⁺ uptake by roots, but did inhibit upward translocation and water transport. Inhibition of photosynthesis and treatments with diethylstilbestrol (10⁻⁵ mol/dm³), as well as with colchicine resulted in inhibition of translocation in plants of low K status, but these inhibitors had little effect on K⁺ uptake by the roots. Oligomycin, 2,4-dinitrophenol and diethylstilbestrol (10⁻⁴ mol/dm³), however, inhibited K⁺ uptake by the roots. In general, K⁺ transport processes were almost unchanged in plants of high K status. It is concluded that only plants of low K status operating with active K⁺ transport mechanisms are responsive to environmental factors. In high K⁺ plants the transport processes are passive and are uncoupled from the metabolic energy flow.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

φX174 lysis requires slyD, a host gene which is related to the FKBP family of peptidyl-prolyl cis-trans isomerases

Abstract: Recessive mutations in the slyD (sensitivity to ly sis) gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage φX174 [1]. The slyD ⁻ mutation, transduced into the normal φX174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function. slyD encodes a protein with 196 residues. A segment corresponding to the first 142 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans isomerases, or rotamases. The C-terminal 46 codons of slyD encode a remarkable histidine-rich peptide which is a metal-binding domain [2]. This sequence is dispensable for slyD function in E -mediated lysis. Although there is no obvious phenotype associated with the slyD ⁻ genotype other than the resistance to E -mediated lysis, overexpression of slyD causes cells to filament and to increase significantly in diameter. Mutations in φX174 can restore the plaque-forming ability of the phage on a slyD ⁻ host. These pos ( p lates on s lyD) mutants plate on E. coli C wild-type and slyD ⁻. A model for SlyD involvement in E function and the role of SlyD in the cell is discussed.