Enhancing anaerobic fermentation of sewage sludge for increasing biogas generation.

The article presents results of biogas generation from sewage sludge after applying two pretreatment methods: sonification and thermal hydrolysis. Original results and literature data of the ultrasonic field influence on biogas generation were compared with literature data concerning effectiveness of the thermal hydrolysis. Sludge pretreatment by the ultrasonic field intensified the biogas production as the amounts of biogas was of ca. 20-24% higher, as compared to the nontreated sludge. The highest generation of biogas was observed after a shorter time. The degree of organic matter reduction was of 45-47%. The content of volatile fatty acids dropped down to 139 mg CH3COOH dm(-3) during 20 days. During the thermal pretreatment of sludge the amounts of generated biogas were ca. 25% higher, as compared to nontreated sludge. The maximum biogas production of 0.92 dm3 was observed in the day 9 of fermentation. To-date results indicate that efficiency of ultrasonic disintegration depends on sonification time, type of heads, as well as power and frequency transmitted to heads.

Ciliary body endophotocoagulation during pars plana vitrectomy for pediatric patients with vitreoretinal disorders and glaucoma.

PURPOSE: To investigate the efficacy of ciliary body endophotocoagulation during pars plana vitrectomy in eyes of pediatric patients with medically uncontrolled glaucoma. METHOD: We compared preoperative and postoperative intraocular pressure and visual acuity in five eyes of five patients aged between 10 months and 14.5 years who had 180 degrees of ciliary body endophotocoagulation with pars plana vitrectomy. RESULTS: Preoperative intraocular pressure on medical therapy for glaucoma ranged from 30 to 55 mm Hg (median, 35 mm Hg). Postoperative follow-up ranged from 12 to 24 months, with a median of 16 months. Postoperative intraocular pressure on medical therapy ranged from 12 to 35 mm Hg (median, 25 mm Hg) at 6 weeks, from 12 to 33 mm Hg (median, 29 mm Hg) at 6 months, and from 12 to 29 mm Hg (median, 27 mm Hg) at 12 months. Twelve months after surgery, mean intraocular pressure reduction of 20 mm Hg was statistically significant (P = .020). CONCLUSION: Ciliary body endophotocoagulation during pars plana vitrectomy may be an effective treatment for pediatric patients with simultaneous uncontrolled glaucoma and vitreoretinal disorders.

Desensitization of beta2-adrenergic receptors with mutations of the proposed G protein-coupled receptor kinase phosphorylation sites.

Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the beta2-adrenergic receptor (betaAR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796-13803). Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407. GRK5 phosphorylated these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites altered desensitization, we have constructed betaARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants betaARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 microM epinephrine treatment of cells. The mutant and wild type (WT) receptors were all desensitized 89-94% after 5 min of 10 microM epinephrine stimulation and 96-98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant betaARs relative to the WT in the extent of 10 microM epinephrine-induced internalization (77-82% after 30 min). Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA- mutant betaARs as well as the WT. We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivo GRK-dependent desensitization of the betaAR.

Salmeterol-induced desensitization, internalization and phosphorylation of the human beta2-adrenoceptor.

1. Partial agonists of the beta2-adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human beta2-adrenoceptors with antagonist. 2. We have compared the activation and desensitization of human beta2-adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS-2B, expressing beta2-adrenoceptor levels of 40-70 fmol mg(-1), and in human embryonic kidney (HEK) 293 cell lines expressing 2-10 pmol mg(-1). The efficacy observed for the stimulation of adenylyl cyclase by salmeterol was only approximately 10% of that observed for adrenaline in BEAS-2B cells expressing low levels of beta2-adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and beta2-adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. 3. The desensitization and internalization of beta2-adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. 4. Our data suggest that the reduction in the rapid phase of desensitization of beta2-adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of beta2-adrenoceptor kinase (betaARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation by these partial agonists appears to be similar to adrenaline.

beta2-adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists.

Previous studies indicated that partial agonists cause less desensitization of the beta2-adrenergic receptor (betaAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of betaAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the betaAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified betaAR behaved identically to the wild type receptor with regard to agonist Kd, coupling efficiency, and desensitization. The coupling efficiencies for betaAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0-30 min) of agonist-induced desensitization of betaAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine >/= fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the betaAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, betaAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of betaAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of betaAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 +/- 0.82) was not appreciably different from ephedrine (5.0 +/- 0.96) and significantly less than that caused by albuterol (10.4 +/- 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of betaARK phosphorylation of the betaAR.

Okadaic acid induces both augmentation and inhibition of beta 2-adrenergic stimulation of cAMP accumulation in S49 lymphoma cells.

To address the role of protein phosphatases in regulating hormonal responses in mammalian cells, we investigated the effects of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, on epinephrine and prostaglandin E1 stimulation of cAMP accumulation and adenylyl cyclase in S49 WT and kin- lymphoma cells. Depending on the dose and time of okadaic acid pretreatment of both cell lines, there were two distinguishable effects on cAMP accumulation, an augmentation and an inhibition. The augmentation occurred rapidly (t1/2 < 1 min), was maximal with 3 microM okadaic acid, and was observed with concentrations of okadaic acid as low as 0.3 microM. Prolonged (t1/2 of 5-15 min) pretreatment of cells with okadaic acid caused an inhibition of epinephrine-stimulated cAMP accumulation, which was characterized by a 2-3-fold increase in the EC50 for the response to epinephrine. The EC50 for the okadaic acid-mediated inhibition was similar to that for the augmentation. In assays of adenylyl cyclase in membrane fractions prepared from okadaic acid-pretreated cells the inhibitory, but not the stimulatory, effects of okadaic acid pretreatment were observed. The data demonstrate that protein phosphatases play an important role in regulating adenylyl cyclase and suggest that cAMP-dependent protein kinase is not involved in either of its actions.

Pathophysiologic correlates of experimental trypanosomiasis in rabbits.

Rabbits inoculated subcutaneously with Trypanosoma brucei brucei developed parasitemia, fever, and reduced food and water intake within 4 to 6 days postinoculation. Subsequent alterations in clinicopathologic parameters included anemia and increased circulating nucleated red blood cells, fibrinogenemia, hypertriglyceridemia, and hyperproteinemia. Transient alterations in the numbers of neutrophils and lymphocytes were detected sporadically; however, leukocytosis was not a characteristic of this chronic infectious condition in rabbits.

Physiological stabilization of rabbits after shipping.

Significant physiological variations that could influence experimental outcomes have been described in laboratory animals following shipping. The objective of the present study was to monitor a variety of physiologic parameters in rabbits after shipping, and to evaluate the time necessary for stabilization of these variables in the new environment. Data indicate that rabbits develop anorexia, hyperglycemia, neutrophilia, lymphopenia and elevated plasma cortisol concentrations immediately after shipping. Most of these effects abate within 2 days after arrival, suggesting that a minimum stabilization period of 48 hours after shipping is advisable prior to use of rabbits in experimental paradigms.