Dephosphorylation of endogenous GABAB receptor R2 subunit and AMPK α subunits which were measured by in vitro method using transfer membrane

Protein phosphorylation can be regulated by changes in kinase activity, phosphatase activity, or both. GABA_B receptor R2 subunit (GABA_BR2) is phosphorylated at S783 by 5′-AMP-activated-protein kinase (AMPK), and this phosphorylation modulates GABA_B receptor desensitization. Since the GABA_B receptor is an integral membrane protein, solubilizing GABA_BR2 is difficult. To circumvent this problem and to identify specific phosphatases that dephosphorylate S783, we employed an in vitro assay based on dephosphorylation of proteins on PVDF membranes by purified phosphatases. Our method allowed us to demonstrate that S783 in GABA_BR2 is directly dephosphorylated by PP2A (but not by PP1, PP2B nor PP2C) in a dose-dependent and okadaic acid-sensitive manner. We also show that the level of phosphorylation of the catalytic subunit of AMPK at T172 is reduced by PP1, PP2A and PP2C. Our data indicate that PP2A dephosphorylates GABA_BR2(S783) less efficiently than AMPK(T172), and that additional phosphatases might be involved in S783 dephosphorylation.     

Extracellular Superoxide Dismutase Induced by Dopamine in Cultured Astrocytes

Under some pathological conditions in brain, a large amount of superoxide anion (O₂ ^?) is produced, causing various cellular damages. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD should play a role to detoxify O₂ ^? in extracellular space; however, a little is known about EC-SOD in brain. Although dopamine (DA) stored in the synaptic vesicle is stable, the excess leaked DA is spontaneously oxidized to yield O₂ ^? and reactive DA quinones, causing damages of dopaminergic neurons. In the present study, we examined the effects of DA on SOD expression in cultured rat cortical astrocytes. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected; however, only EC-SOD was increased by DA exposure for 24 h, dose-dependently. The expression of EC-SOD protein and the cell-surface SOD activity in astrocytes also increased with 100 μM DA exposure. The increase of EC-SOD mRNA by DA was inhibited by a DA transporter inhibitor, GBR12909, whereas it was not changed by DA receptor antagonists, SKF-83566 (D1) and haloperidol (D2). Furthermore, a monoamine oxidase inhibitor, pargyline, and antioxidants, N-acetyl-l-cysteine and glutathione, also did not affect the DA-induced expression of EC-SOD mRNA. On the other hand, an inhibitor of nuclear factor kappaB (NF-κB), ammonium pyrrolidine-1-carbodithioate, suppressed the DA-induced expression of EC-SOD mRNA. These results suggest that DA incorporated into the cells caused the induction of EC-SOD mRNA followed by the enhancements of EC-SOD protein level and the enzyme activity, and that NF-κB activation is involved in the mechanisms of the EC-SOD induction. The regulation of EC-SOD in astrocytes surrounding dopaminergic neurons may contribute to the defensive mechanism against oxidative stress in brain.     

On the diet of the Pharaoh Eagle Owl,Bubo ascalaphus (Savigny, 1809), in Qatar,with an overview of its feeding habits

The diet of Bubo ascalaphus in Qatar was assessed based on pellets collected from the first known nesting site of the species in the country. The pellets contained a total of 68 prey items, representing 9 different species: 4 mammals, 1 bird, 1 reptile, and at least 3 scorpions. Mammals clearly comprised the major food source (89.7% and 97.7% in frequency and biomass respectively). Our data suggest that Pharaoh Eagle Owls are opportunistic predators that feed on a variety of prey depending on their temporal/spatial availability, which is consistent with previous studies. A literature review clearly suggests that Eagle Owls in arid to semi-arid environments are opportunistic predators with small mammals being their main prey. Predation on migrating Blue-cheeked Bee-eaters Merops persicus supports this hypothesis.     

Characterization of Acid-Cyclodextrin Glycosyltransferase of an Alkalophilic Bacillus sp.

Three kinds of lectins (LOL-I, II and III) were isolated from seeds of Lathyrus odoratus (sweet pea) in a homogenous form. The three fractions agglutinated the erythrocytes of laying hens, and the agglutination was strongly inhibited by α-methyl d-mannoside and d-mannose. However, they did not agglutinate those of the males and nonlyaing hens, differing from concanavalin A which showed a similar binding specificity for monosaccharide to LOL and agglutinated all types of erythrocytes derived from chicken in this study. LOL–I and II had a molecular weight of 52,000 and both consisted of two large (20,000 daltons) and two small subunits (6000 daltons). LOL–III had a molecular weight of 55,000, and its subunit structure was different from those of LOL–I and II. The amino acid compositions of the three fractions were very similar. They contained large amounts of aspartic acid, threonine, serine and valine, but no cysteine or methionine. Circular dichroism measurements indicated that β-structure was a major secondary structure of these lectins. The addition of α-methyl d-mannoside or d-mannose had significant effects on the CD spectra in the near-ultraviolet region, but no detectable change was observed in the 200~250 nm region. LOL–I had two binding sites for d-mannose, and the association constant was about 1000 liters per mol.     

Purification and Properties of Neutral-cyclodextrin Glycosyl-transferase of an Alkalophilic Bacillus sp.

Neutral-cyclodextrin glycosyltransferase (EC 3.2.1.19) of alkalophilic Bacillus sp. (ATCC 21783) was purified by starch adsorption, DEAE-cellulose chromatography and Sephadex G–150 gel filtration chromatography followed by preparative polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was 85,000-88,000 by SDS-disc gel electrophoresis. The enzyme was most active at pH 7 and 50°C, and stable up to 60°C at pH 7 and in the range of pH 6~8 at 60°C by 30 min incubation. The apparent V_max and Km values for α- and β-cyclodextrin at a constant concentration of sucrose were 417, 70 µmoles glucose/min · mg protein and 10, 0.83 nm, respectively. About 85~90% of amylose, 75~80% of potato starch, 65~70% of amylopectin, 55~60% of glycogen, 45~50% of amylopectin β-limit dextrin, 20~25% of maltotriose and 10~15% of maltose were converted to cyclodextrins with 0.5~1% (w/v) of each substrate.

Schardinger β-dextrin was preferentially produced from starch, and α- or γ-dextrin was gradually formed after prolonged incubation. After 20 min incubation, about 0.4, 14 and 2.5% of α-, β- and γ-dextrin were formed from starch, respectively.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2′-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27 and p18 in endocrine organs

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27^Kip1/CDKN1B (p27) and p18^Ink4C/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.     

Genetic ablation and chemical inhibition of IP3R1 reduce mutant huntingtin aggregation

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Previously, it has been shown that inhibition of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) activity reduced aggregation of pathogenic polyQ proteins. Experimentally, this effect was achieved by modification of the intracellular IP3 levels or by application of IP3R1 inhibitors, such as 2-aminoethyl diphenylborinate (2-APB). Unfortunately, there are certain concerns about the 2-APB specificity and cytotoxicity. Moreover, a direct link between IP3R1 and polyQ aggregation has not been shown yet. In this study we show, that down-regulation of the IP3R1 levels by shRNA reduced the aggregation of mutant htt. We tested 2-APB analogs in an attempt to identify less toxic and more IP3R1-specific compounds and found that the effect of these analogs on the reduction of the mutant htt aggregation did weakly correlate with their inhibitory action toward the IP3-induced Ca(2+) release (IICR). Their effect on aggregation was not correlated with the store-operated Ca(2+) entry (SOCE), which is another target of the 2-APB related compounds. Our findings suggest that besides functional contribution of the IP3R inhibition on the mutant htt aggregation there are additional mechanisms for the anti-aggregation effect of the 2-APB related compounds.