Effects of glutamine,methionine sulfone and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells

Glutamine synthetase (EC 6.3.1.2) activity of hepatoma tissue culture cells is elevated by cortocisteroids and depressed by glutamine (Kulka, R.G., Tomkins, G.M. and Crook, R.B. (1972) J. Cell Biol., 54, 175–179). The transfer of cells from high (1–5 mM) to low (0.2–0.4 mM) concentrations of glutamine causes a marked increase in glutamine synthetase activity. The addition of a glutamine antagonist, methionine sulfone (1 mM) to cells suspended in high (1 mM) concentrations of glutamine also causes an increase of glutamine synthetase activity which is greater than that elicited by the transfer of cells to low concentrations of glutamine. Rates of synthesis of glutamine synthetase have been measured by radioimunoprecipitation in hepatoma tissue culture cells incubated under various conditions. Incubation of cells with the synthetic corticosteroid hormone, dexamethasone, markedly stimulates the relative rate of glutamine synthetase biosynthesis. Glutamine, or its analogue, methionine sulfone, have no effect on the relative rate of synthesis of the enzyme. However, total protein and RNA synthesis increase markedly with increasing external glutamine concentration in the range 0–1 mM. Methionine sulfone (1 mM) inhibits the degradation of glutamine synthetase in the presence of 1 mM glutamine. The data are consistent with the conclusion that the corticosteroid, dexamethasone, elevates glutamine synthetase activity by stimulating its rate of synthesis, whereas methionine sulfone elevates glutamine synthetase activity by inhibiting the glutamine-stimulated degradation of preformed enzyme.     

Tyrosine Phosphorylation of Protein Kinase Cδ Is Essential for Its Apoptotic Effect in Response to Etoposide

Protein kinase Cdelta (PKCdelta) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCdelta in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G(1)/S phase of the cell cycle. Overexpression of PKCdelta increased the apoptotic effect induced by etoposide, whereas the PKCdelta selective inhibitor rottlerin and the PKCdelta dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCdelta and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCdelta were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCdelta in the effects of etoposide was examined using cells overexpressing a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCdelta. Likewise, activation of caspase 3 and the cleavage of the PKCdelta5 mutant were significantly lower in cells overexpressing PKCdelta5. Using mutants of PKCdelta altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCdelta in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCdelta as an important regulator of this effect.     

Histone modifications associated with drought tolerance in the desert plant <Emphasis Type="Italic">Zygophyllum dumosum</Emphasis> Boiss

Zygophyllum dumosum Boiss. is a perennial Saharo-Arabian phytogeographical element and a dominant shrub on the rocky limestone southeast-facing slopes of the Negev desert. The plant is highly active during the winter, and semideciduous during the dry summer, i.e., it sheds its leaflets, while leaving the thick, fleshy petiole green and rather active during the dry season. Being resistant to extreme perennial drought, Z. dumosum appears to provide an intriguing model plant for studying epigenetic mechanisms associated with drought tolerance in natural habitats. The transition from the wet to the dry season was accompanied by a significant decrease in nuclear size and with posttranslational modifications of histone H3 N-terminal tail. Dimethylation of H3 at lysine 4 (H3K4)—a modification associated with active gene expression—was found to be high during the wet season but gradually diminished on progression to the dry season. Unexpectedly, H3K9 di- and trimethylation as well as H3K27 di- and trimethylation could not be detected in Z. dumosum; H3K9 monomethylation appears to be prominent in Z. dumosum during the wet but not during the dry season. Contrary to Z. dumosum, H3K9 dimethylation was detected in other desert plants, including Artemisia sieberi, Anabasis articulata and Haloxylon scoparium. Taken together, our results demonstrate dynamic genome organization and unique pattern of histone H3 methylation displayed by Z. dumosum, which could have an adaptive value in variable environments of the Negev desert.     

The effects of breathing pattern training on ventilatory function in patients with COPD

The purpose of this study was to assess the effects of a particular breathing pattern training (BPT) on forced expiratory volume during the first second (FEV₁) and forced vital capacity (FVC) in patients with chronic obstructive pulmonary disease (COPD). The subjects adjusted each breath to a target breath displayed on a video screen, by using visual feedback. This target was chosen in an individual sample recorded at rest. We used a randomized, controlled group design. Twenty patients with stable COPD, FEV₁ less than 1.5 liters, undergoing a traditional rehabilitation program were randomly assigned to the BPT group or to the control group. Each BPT subject underwent 30–35 training sessions spread out over four weeks, in addition to the traditional program. FEV₁ and FVC were performed before and after this program. ANOVAs showed that FEV₁ and FVC significantly improved in BPT subjects, with a mean percent increase of 22% and 19%, respectively. Corresponding changes in controls were not significant. This study showed short-term increases in FEV₁ and FVC in COPD patients practicing BPT in addition to respiratory rehabilitation, in comparison with controls. Further studies should incorporate outcome data to clarify the mechanisms and the duration of this effect.The authors express their gratitude to Philippe Carrias, Elizabeth Maclet, Françoise Tulane, and Bernard Cossalter for their contribution to this study, as well as to Michèle Delaire for her technical assistance.     

High yellow pigments production by root culture of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and its release in medium under gas oil treatment

In this study, the ability of safflower-isolated root cultures to produce yellow pigments was tested. Initially, the growth of isolated roots in static liquid medium was evaluated with different volumes of culture medium. A volume of 6 ml of medium per flask of 250 ml gave the best growth performance and, in this condition of culture, production of pigments from isolated roots treated or not by light has been determined by spectrophotometry (321 and 400 nm). Under these conditions, the production of yellow pigments amounted to 13.18 mg g⁻¹ fresh weight and the light stimulated the synthesis of these pigments by isolated roots. Total yellow pigments of 24.12, 38.91 and 46.38 mg g⁻¹ fresh weight was produced by the roots treated with 9, 13.5 and 18% (v/v) gas oil, respectively, representing high values of production. The pigments were released in large quantities in the medium. The increased synthesis of pigments as a result of gas oil treatment was accompanied by a reduction of the peroxidase activity of roots. Given the high production of yellow pigments, systems of isolated root culture could be considered for the study of a larger scale production of safflower pigments widely used for various industrial purposes.     

The activity of 2,5-oligoadenylate synthetase, an interferon-induced enzyme, is coupled to the differentiation state of mouse condylar cartilage

The enzyme 2,5-oligoadenylate synthetase (2-5A synthetase) is associated with the interferon system, with special reference to the differentiation process of various cell types. The present study investigated whether 2-5A synthetase is also involved in the differentiation of neocartilage in perinatal mice. The cartilage of the mandibular condyle, a secondary type of cartilage, develops relatively late in prenatal life; and consequently it was possible to obtain a relatively embryonic cartilage at a developmental stage that could be manipulated enzymatically, in order to separate and thereby obtain its undifferentiated, proliferative portion along with its more mature fraction. Immunohistochemical studies using antibodies against type I and type II collagen and cartilage-specific proteoglycans could have determined the differentiation status of various portions of the developing condyle. However, the above methodology lacks the necessary precision and accuracy to indicate subtle changes in cellular differentiation. It became evident that the activity of 2-5A synthetase was indeed different in cellular compartments that were at different stages of differentiation. In the neonatal condyle the highest level of activity was encountered in proliferating and as yet undifferentiated prechondrocytes, whereas fully differentiated chondrocytes showed a marked decrease in the activity of this enzyme.