Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5–96 h) with [³H]galactose, [³H]glucosamine, or [³H]myoinositol and treatment of the purified [³H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([³H]galactose and [³H]myoinositol) or 72 h ([³H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([³H]galactosamine, [³H]mannose, and [³H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5–72 hr) with [¹⁴C]linoleate, [³H]myristate, [³H]-oleate, [³H]palmitate, or [³H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [³H]palmitate and [³H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA₂ treatment of the dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [³H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 μg/ml), or the membrane permeable cAMP analog (but)₂ cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)₂ cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [³H]glucosamine in the presence of (but)₂cAMP (1 mM), TPA (10^?7 M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [³H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone. In cells differentiated with FSH (30 ng/ml for 3 days) to induce LH receptors, treatment with hCG (100 ng/ml) induced a rapid (60 sec) and transient (5 min) decrease in the GPI content, whereas no efect of the hormone on undifferentiated granulosa cells could be observed. The rapid effect elicited by hCG on GPI content and turnover may be an early transduction mechanism involved in the biological effects of LH/hCG in differentiated granulosa cells. © 1993 Wiley-Liss, Inc.     

Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss mouse 3T3 cells

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F_2α alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF_2α stimulation. In contrast tunicamicin does not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF_2α     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Creation of an allosteric phosphofructokinase starting with a nonallosteric enzyme. The case of dictyostelium discoideum phosphofructokinase.

An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n(H) = 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S(0.5) = 4500 microm), strong activation by fructose 2,6-bisphosphate (K(act) = 0.1 microm) and fructose 1,6-bisphosphate (K(act) = 40 microm), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n(H) = 1.0; K(m) = 22 microm) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu(834), as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.     

Mouse Ganglion-Cell Photoreceptors Are Driven by the Most Sensitive Rod Pathway and by Both Types of Cones

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are depolarized by light by two mechanisms: directly, through activation of their photopigment melanopsin; and indirectly through synaptic circuits driven by rods and cones. To learn more about the rod and cone circuits driving ipRGCs, we made multielectrode array (MEA) and patch-clamp recordings in wildtype and genetically modified mice. Rod-driven ON inputs to ipRGCs proved to be as sensitive as any reaching the conventional ganglion cells. These signals presumably pass in part through the primary rod pathway, involving rod bipolar cells and AII amacrine cells coupled to ON cone bipolar cells through gap junctions. Consistent with this interpretation, the sensitive rod ON input to ipRGCs was eliminated by pharmacological or genetic disruption of gap junctions, as previously reported for conventional ganglion cells. A presumptive cone input was also detectable as a brisk, synaptically mediated ON response that persisted after disruption of rod ON pathways. This was roughly three log units less sensitive than the rod input. Spectral analysis revealed that both types of cones, the M- and S-cones, contribute to this response and that both cone types drive ON responses. This contrasts with the blue-OFF, yellow-ON chromatic opponency reported in primate ipRGCs. The cone-mediated response was surprisingly persistent during steady illumination, echoing the tonic nature of both the rod input to ipRGCs and their intrinsic, melanopsin-based phototransduction. These synaptic inputs greatly expand the dynamic range and spectral bandpass of the non-image-forming visual functions for which ipRGCs provide the principal retinal input.