Identification of Staphylococcus aureus and Escherichia coli isolated from Egyptian food by conventional and molecular methods

Staphylococcus aureus and Escherichia coli were enumerated and isolated from ready-to-eat vegetables salad and meat luncheon on their selective media (Baird-parker and Macconkey agar, respectively). Twenty suspicious colonies of each (10 from each product) were randomly chosen and identified using conventional based on morphological and physiological characteristics. S. aureus and E. coli isolates which gave the highest pathogenicity were chosen for identification and confirmation with molecular method based on 16S rRNA gene. The PCR amplification method of 16S rRNA gave the same identification results as conventional method, but it was sensitive and fast. This molecular method takes about 48 h in comparison with 6 days for conventional method. The 16S rRNA of S. aureus and E. coli were deposited in the Genebank database under accessions (AB599719.1 and AB599716.1, respectively).     

Alcohol-associated folate disturbances result in altered methylation of folate-regulating genes

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The steady-state accumulation of folate seems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma-glutamyl hydrolase (GGH), which removes them, enabling it to be transported across the biological membranes. Overexpression of GGH and downregulation of FPGS would be expected to decrease intracellular folate in its polyglutamylated form, thereby increasing efflux of folate and its related molecules, which might lead to resistance to drugs or folate deficiency. The study was sought to delineate the activity of GGH and expression FPGS in tissues involved in folate homeostasis during alcoholism and the epigenetic regulation of these enzymes and transporters regulating intracellular folate levels. We determined the activity of GGH and expression of FPGS in tissues after 3 months of ethanol feeding to rats at 1 g/kg body weight/day. The results showed that there was not any significant change in the activity of folate hydrolyzing enzyme GGH in ethanol-fed rats while there was significant down regulation in the expression of FPGS. Ethanol feeding decreased the total as well as polyglutamated folate levels. There was tissue-specific hyper/hypo methylation of folate transporter genes viz. PCFT and RFC by chronic ethanol feeding. Moreover, hypermethylation of FPGS gene was observed in intestine and kidney without any change in methylation levels of GGH in the ethanol-fed rats. In conclusion, the initial deconjugation of polyglutamylated folate by GGH was not impaired in ethanol-fed rats while the conversion of monoglutamylated folate to polyglutamylated form might be impaired. There was tissue-specific altered methylation of folate transporter genes by chronic ethanol feeding.     

Inhibition of gut- and lung-derived serotonin attenuates pulmonary hypertension in mice

Decreasing the bioavailability of serotonin (5-HT) by inhibiting its biosynthesis may represent a useful adjunctive treatment of pulmonary hypertension (PH). We assessed this hypothesis using LP533401, which inhibits the rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) expressed in the gut and lung, without inhibiting Tph2 expressed in neurons. Mice treated repeatedly with LP533401 (30-250 mg/kg per day) exhibited marked 5-HT content reductions in the gut, lungs, and blood, but not in the brain. After a single LP533401 dose (250 mg/kg), lung and gut 5-HT contents decreased by 50%, whereas blood 5-HT levels remained unchanged, suggesting gut and lung 5-HT synthesis. Treatment with the 5-HT transporter (5-HTT) inhibitor citalopram decreased 5-HT contents in the blood and lungs but not in the gut. In transgenic SM22-5-HTT+ mice, which overexpress 5-HTT in smooth muscle cells and spontaneously develop PH, 250 mg/kg per day LP533401 or 10 mg/kg per day citalopram for 21 days markedly reduced lung and blood 5-HT levels, right ventricular (RV) systolic pressure, RV hypertrophy, distal pulmonary artery muscularization, and vascular Ki67-positive cells (P < 0.001). Combined treatment with both drugs was more effective in improving PH-related hemodynamic parameters than either drug alone. LP533401 or citalopram treatment partially prevented PH development in wild-type mice exposed to chronic hypoxia. Lung and blood 5-HT levels were lower in hypoxic than in normoxic mice and decreased further after LP533401 or citalopram treatment. These results provide proof of concept that inhibiting Tph1 may represent a new therapeutic strategy for human PH.     

Stilbene glucosides from the bulbs of Iris tingitana

Two new dimeric stilbene glucosides, tingitanol A (1) and tingitanol B (2) together with trans-resveratrol 3-O-glucopyranoside (3) in addition to three known isoflavones, 5-O-methylgenistein (4), 5-O-methylgenistein 7-O-β-d-glucopyranoside (5) and betavulgarin (6) have been isolated for the first time from the fresh bulbs of Iris tingitana Boiss. & Reut. Their structures were established on the basis of the spectral data and direct comparison with values from previously identified analogues. Additionally, the isolated compounds (1–6) were evaluated for the free radical scavenging activity.     

Different feeding strategy for the production of biosurfactant from Pseudomonas aeruginosa USM AR2 in modified bioreactor

A locally-isolated Pseudomonas aeruginosa USM AR2 possessing the ability to produce glycolipid-type biosurfactant (rhamnolipid) was used in this research to explore fermentation technology for rhamnolipid production. Rhamnolipid concentration in 2.5 L fed-batch fermentation was improved from 0.173 to 8.06 g/L by manipulating the feeding strategy and cultivation protocol. The culture was fed with petroleum diesel and complex medium. The highest rhamnolipid concentration was achieved when the culture was initially fed with both petroleum diesel and complex medium, followed by feeding of petroleum diesel only at the end of the stationary phase. Severe foaming problem was resolved by modifying and integrating a foam recycler to the bioreactor. This successfully extended the cultivation period and increased the yield of final rhamnolipid. No antifoam agent was added as this modified bioreactor allowed cultivation to proceed even under foam generation. The viscosity measurement, surface tension activity test, and drop-collapse test were performed as an indirect measure of rhamnolipid presence.     

Alcohol and PRAS40 knockdown decrease mTOR activity and protein synthesis via AMPK signaling and changes in mTORC1 interaction

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GβL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS‐1. EtOH also caused changes in mTORC1 protein–protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14‐3‐3 to raptor, while the PRAS40 and 14‐3‐3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14‐3‐3, whereas decreased GβL–mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GβL with mTOR, while likewise increasing the interaction of raptor with 14‐3‐3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes. J. Cell. Biochem. 109: 1172–1184, 2010. © 2010 Wiley‐Liss, Inc.     

Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies

A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 µm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non‐functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q_máx of 344 mg IgG/g support and K_a of 1.5 × 10⁵ M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine‐NaOH (pH 11) buffer. Copyright © 2010 John Wiley & Sons, Ltd.