The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

Anti-semaphorin 3A antibodies rescue retinal ganglion cells from cell death following optic nerve axotomy

Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961-971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2-3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A-derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death.     

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum

Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.     

Effects of training on the exercise-induced changes in serum amino acids and hormones

The purpose of this study was to examine power-type athletes to determine changes in amino acid and hormone concentrations in circulating blood following 2 different high-intensity exercise sessions before and after the 5-week training period. Eleven competitive male sprinters and jumpers performed 2 different running exercise sessions: a short run session (SRS) of 3 x 4 x 60 m (intensity of 91-95%) with recoveries of 120 and 360 seconds, and a long run session (LRS) with 20-second intervals (intensity of 56-100%) with recoveries of 100 seconds to exhaustion. The concentrations of serum amino acids, hormones, and lactate were determined from the blood samples drawn after an overnight fast and 10 minutes before and after both SRS and LRS. The average blood lactate concentrations were 12.7 +/- 1.6 mmol;pdL(-1) and 16.6 +/- 1.4 mmol;pdL(-1) (p < 0.01) following SRS and LRS, respectively. The average total running time was longer (p < 0.001) following LRS (164 +/- 20 seconds) than following SRS (91 +/- 8 seconds). The fasting levels of all amino acids decreased (p = 0.024; 19.4%) after the 5-week period, whereas an increase (p = 0.007; 24.5%) was observed in the fasting concentration of testosterone (TE). The exercise sessions induced no changes in the total sum of all amino acids, but significant increases or decreases were observed in single amino acids. When the range of the relative concentration changes before and after the training period was compared, significant decreases were found in valine (p = 0.048), asparagine (p = 0.029), and taurine (p = 0.030) following SRS. There were significant increases in the absolute hormonal concentration changes following LRS with TE (p = 0.002; 30.4%), cortisol (COR; p = 0.006; 12.0%), and in the TE/COR ratio (p = 0.047; 21.0%) but not in the concentration of growth hormone (GH). The results of the study indicate that the speed and strength training period strongly decreases the fasting concentrations of amino acids in the power-trained athletes in a good anabolic state with the daily protein intake of 1.26 g;pdkg(-1) body weight. At the same time the intensive lactic exercise session induces strong decreases, especially in valine, asparagine, and taurine.