Characterization of the proteolytic compartment in rat hepatocyte nodules

Persistent liver nodules (hepatocyte nodules, neoplastic nodules) were produced in rat liver by intermittent feeding with 2-acetylaminofluorene. Dense bodies (secondary lysosomes) were purified and characterized from the nodules. The purity of the dense body fraction was 90%. The levels of various lysosomal enzyme activities were lower in these dense bodies in comparison with dense bodies from control liver. Similarly, protein degradation was 50% lower in dense bodies from liver nodules than in control liver. The number of autophagic vacuoles (AVs) in the nodular tissue increased considerably after 3 h vinblastine treatment. We have taken advantage of this expansion in an effort to isolate these organelles from liver nodules. Autophagic vacuoles have been isolated recently from liver and kidney but not from putatively premalignant liver nodules. Fraction purity of AVs from liver nodules was 95%. As with dense bodies, AVs from nodular tissue displayed lower activities of proteinases and lower rates of protein degradation when compared with their counterparts from normal liver tissue. Accordingly, the lower rate of overall protein degradation in liver nodules can be ascribed to a decrease in lysosomal activity. A diminished autophagic sequestration capacity is the most plausible explanation for the decreased rate of proteolysis in cells. This could conceivably give these nodular cells a growth advantage and assist in their selective outgrowth as well as in their transformation from neoplastic into true cancer cells.     

N‐acetyl‐L‐cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors

Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N‐acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA‐2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase‐3, ‐8, ‐9 activities and Bcl‐2, Bax, Cyt‐c, Annexin V‐FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD₅₀) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD₅₀) and H₂O₂ for 24 h increased Caspase‐3, ‐8, ‐9 activities, Cyt‐c and Bax levels and decreased Bcl‐2 levels. The concurrent incubation of NT2 cells with bleomycin/H₂O₂ and NAC (5 mM) for 24 h abolished bleomycin/H₂O₂‐dependent increases in Caspase‐3, ‐8, ‐9 activities, Bax and Cyt‐c levels and bleomycin/H₂O₂‐dependent decrease in Bcl‐2 level. Our results indicate that bleomycin/H₂O₂ induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H₂O₂ induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin‐induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells. J. Cell. Biochem. 114: 1685–1694, 2013. © 2013 Wiley Periodicals, Inc.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemagglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected.     

Importance of pfkA for rapid growth of Enterobacter cloacae during colonization of crop seeds

Enterobacter cloacae A-11 is a prototrophic, glycolytic mutant of strain 501R3 with a single transposon insertion in pfkA. The populations of strain A-11 on cucumber and radish seeds were smaller than the populations of strain 501R3 in natural soil, but the populations of these two strains on pea, soybean, sunflower, and sweet corn seeds were similar (D. P. Roberts, P. D. Dery, I. Yucel, J. Buyer, M. A. Holtman, and D. Y. Kobayashi, Appl. Environ. Microbiol. 65:2513-2519, 1999). The net effect of the mutation in pfkA in vitro was a shift from rapid growth on certain carbohydrates detected in seed exudates to much slower growth on other carbohydrates, amino acids, and organic acids. The impact of the mutation in pfkA was greatest on the growth rate of E. cloacae on the seeds that released the smallest quantities of fructose, other carbohydrates, and amino acids. Corn, pea, soybean, and sunflower seeds released total amounts of carbohydrates and amino acids at rates that were approximately 10- to 100-fold greater than the rates observed with cucumber and radish seeds for the first 24 h after inhibition began. The growth rate of strain A-11 was significantly less (50% less) than the growth rate of strain 501R3 on radish seeds, and the growth rate of strain A-11 was too low to estimate on cucumber seeds in sterile sand for the first 24 h after inhibition began. The growth rate of strain A-11 was also significantly lower on soybean seeds, but it was only 17% lower than the growth rate of strain 501R3. The growth rates of strains 501R3 and A-11 were similar on pea, sunflower, and corn seeds in sterile sand for the first 30 h after imbibition began. Large reductions in the growth rates of strain A-11 on seeds were correlated with subsequent decreased levels of colonization of seeds compared to the levels of colonization of strain 501R3. The strain A-11 populations were significantly smaller than the strain 501R3 populations only on radish and cucumber seeds. The mutation in pfkA appears to decrease the level of colonization by E. cloacae for seeds that release small quantities of reduced carbon compounds by decreasing the size of the pool of compounds that support rapid growth by this bacterium.     

Drought-induced oxidative damage and antioxidant responses in peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) seedlings

Two cultivars of peanut (Arachis hypogaea L.) which were designated as resistant (Florispan) and sensitive (Gazipasa) according to their growth retardation under drought stress conditions were compared for their oxidative damage and antioxidant responses. Sixteen days-old peanut seedlings were subjected to PEG-6000 solutions of two different osmotic potentials; −0.4 and −0.8 MPa, and various growth parameters, photosystem II activity, changes in malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline levels, activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POX) and gluthatione reductase (GR) enzymes were determined. Both cultivars exhibited water deficit at −0.8 MPa osmotic potential of PEG-6000 and H₂O₂ levels significantly increased during exposure to −0.4 MPa osmotic potential. However, H₂O₂ levels were under control in both cultivars at exposure to −0.8 MPa osmotic potential. Significant proline accumulation was observed in the tissues of cv. Florispan at −0.8 MPa osmotic potential, whereas proline accumulation did not appear to be an essential part of the protection mechanism against drought in cv. Gazipasa. No significant variation in chlorophyll fluorescence values were detected in neither of the cultivars. Enzyme activity measurements revealed that Gazipasa copes well with lesser magnitudes of drought stress by increasing the activity of mainly APX, and during harsh stress conditions, only APX maintains its activity in the tissues. In cultivar Florispan, GR activity appears to take role in lesser magnitudes of drought stress, whereas CAT and APX activities appear to be very crucial antioxidative defenses during intense stress conditions. The results indicate that, the level of proline and activities of the enzymes CAT and APX are important mechanisms for the maintenance of drought tolerance in peanut plants.     

Formulation and <Emphasis Type="Italic">In vitro</Emphasis> Characterization of Eudragit® L100 and Eudragit® L100-PLGA Nanoparticles Containing Diclofenac Sodium

The aim of this study was to formulate and characterize Eudragit® L100 and Eudragit® L100-poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing diclofenac sodium. Diclofenac generates severe adverse effects with risks of toxicity. Thus, nanoparticles were prepared to reduce these drawbacks in the present study. These nanoparticles were evaluated for surface morphology, particle size and size distribution, percentage drug entrapment, and in vitro drug release in pH 6.8. The prepared nanoparticles were almost spherical in shape, as determined by atomic force microscopy. The nanoparticles with varied size (241–274 nm) and 25.8–62% of entrapment efficiency were obtained. The nanoparticles formulations produced the release profiles with an initial burst effect in which diclofenac sodium release ranged between 38% and 47% within 4 h. The extent of drug release from Eudragit® L100 nanoparticles was up to 92% at 12 h. However, Eudragit®/PLGA nanoparticles showed an initial burst release followed by a slower sustained release. The cumulative release at 72 h was 56%, 69%, and 81% for Eudragit®/PLGA (20:80), Eudragit®/PLGA (30:70) and Eudragit®/PLGA (50:50) nanoparticles, respectively. The release profiles and encapsulation efficiencies depended on the amount of Eudragit in the blend. These data demonstrated the efficacy of these nanoparticles in sustaining the diclofenac sodium release profile.     

Sleep disturbances and excessive daytime sleepiness in migraine: A comparison between comorbidities and disability

Sleep and Biological Rhythms - Many studies have investigated the association between headache and sleep disorders, but few have focused on migraine. The goal of this study was to evaluate sleep...