Lipocalin 2 regulation by thermal stresses: Protective role of Lcn2/NGAL against cold and heat stresses

Environmental temperature variations are the most common stresses experienced by a wide range of organisms. Lipocalin 2 (Lcn2/NGAL) is expressed in various normal and pathologic conditions. However, its precise functions have not been fully determined. Here we report the induction of Lcn2 by thermal stresses in vivo, and its role following exposure to cold and heat stresses in vitro. Induction of Lcn2 in liver, heart and kidney was detected by RT-PCR, Western blot and immunohistochemistry following exposure of mice to heat and cold stresses. When CHO and HEK293T cells overexpressing NGAL were exposed to cold stress, cell proliferation was higher compared to controls. Down-regulatrion of NGAL by siRNA in A549 cells resulted in less proliferation when exposed to cold stress compared to control cells. The number of apoptotic cells and expression of pro-apoptotic proteins were lower in the NGAL overexpressing CHO and HEK293T cells, but were higher in the siRNA-transfected A549 cells compared to controls, indicating that NGAL protects cells against cold stress. Following exposure of the cells to heat stress, ectopic expression of NGAL protected cells while addition of exogenous recombinant NGAL to the cell culture medium exacerbated the toxicity of heat stress specially when there was low or no endogenous expression of NGAL. It had a dual effect on apoptosis following heat stress. NGAL also increased the expression of HO-1. Lcn2/NGAL may have the potential to improve cell proliferation and preservation particularly to prevent cold ischemia injury of transplanted organs or for treatment of some cancers by hyperthermia.     

Protein complex prediction based on <Emphasis Type="Italic">k</Emphasis>-connected subgraphs in protein interaction network

Background  

Protein complexes play an important role in cellular mechanisms. Recently, several methods have been presented to predict protein complexes in a protein interaction network. In these methods, a protein complex is predicted as a dense subgraph of protein interactions. However, interactions data are incomplete and a protein complex does not have to be a complete or dense subgraph.     

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 Å crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 Å out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid’s origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.     

Using the Modified Lee Model Code to Calculate the Optimum Length of NiZn Ferrite to Enhance the DPF Argon SXR Emission

Plasma Physics Reports - A modified version of the Lee Model Code with better calculation of the magnetic field inside the Dense Plasma Focus (DPF) tube has been used to investigate the NiZn...     

Enzymatic hydrolysis of racemic ibuprofen esters using Rhizomucor miehei lipase immobilized on different supports

This research describes the immobilization of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH₂-RML) on different supports including octyl-sepharose (octyl-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH₂-RML), glyoxyl sepharose (Gx-RML and Gx-NH₂-RML) and glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH₂-RML). The highest immobilization yield was achieved for octyl-RML (>98%) followed by CNBr-RML (88%). Octyl-RML had the most specific activity (13.6) among all derivatives. The other preparations had moderate activities likely because of chemical reaction during covalent attachment of the enzyme. The catalytic behavior of lipase immobilized in hydrolysis reactions was investigated using methyl, ethyl, propyl, butyl and isobutyl-ibuprofen esters and the influence of the alkyl chain and the alcoholic residue of the ester were studied. Butyl ester was the most interesting ester for carrying out hydrolysis. The highest enantioselectivity of enzyme (E = 8.8) was obtained with isooctane/sodium phosphate buffer pH 7.0 at temperature of 40 °C. Increasing temperature from 40 to 50 °C caused decreasing in enantioselectivities and conversions. Also esterification of ibuprofen was carried out in solvent systems containing isooctane and two ionic liquids (ILs); [BMIM][PF₆] and [BMIM][BF₄]. Poor conversions and enantioselectivities were observed during esterification in all solvents.     

Efficiency of Plasmocin™ on various mammalian cell lines infected by mollicutes in comparison with commonly used antibiotics in cell culture: a local experience

Mycoplasma contamination is a deleterious event for cell culture laboratories. Plasmocin™ is used to prevent and eradicate mycoplasma infections from cell. In this study, 80 different mammalian cell lines from various sources; human, monkey, mice, hamster and rat were used to study and evaluate plasmocin™ efficiency and compare it to commonly used antibiotics such as BM-cyclin, ciprofloxacin and mycoplasma removal agent (MRA). It was shown that mycoplasma infections were eradicated by plasmocin™, BM-cyclin, ciprofloxacin and MRA in 65%, 66.25%, 20%, and 31.25%, respectively, of infected cell cultures. However, re-infection with mycoplasmas after the period of 4 months occurred in 10–80% of the studied cell lines. Cell cytotoxicity and culture death was observed in 25, 17.5 and 10% of the treated cells, for plasmocin™, BM-cyclin and MRA, respectively. In this study, Plasmocin™ showed strong ability to eradicate mollicutes from our cell lines with minimal percentage of regrowth. However, due to its high cell cytotoxicity it should be used with caution especially when dealing with expensive or hard-to-obtain cell lines. Amongst the antibiotics tested, BM-cyclin was shown to remove mycoplasma with the highest efficiency.     

Neutrophil gelatinase-associated lipocalin: a new antioxidant that exerts its cytoprotective effect independent on Heme Oxygenase-1

Neutrophil Gelatinase-Associated Lipocalin (NGAL/Lcn2), a member of the lipocalin family, has a variety of functions. There are extensive studies examining the expression of NGAL under harmful conditions. However, its precise function remains poorly understood. Heme Oxygenase 1 (HO-1) is an enzyme with well-established cytoprotective effects. Previous work showed that NGAL induces expression of HO-1. Interestingly, the same stimuli induced the expression of both NGAL and HO-1. The current study was designed to (1) determine whether NGAL exerts its cytoprotective effect through HO-1 and (2) compare NGAL and HO-1 with each other in terms of their protective role against oxidative stress. The current data indicate that NGAL exerts its cytoprotective effect independent of HO-1 and protects cells against oxidative stress more efficiently than HO-1. The data also strongly suggest that induction of NGAL under harmful conditions is a compensatory response to ameliorate oxidative stress-mediated toxicity. These findings may suggest new applications of NGAL, particularly when oxidative stress is a major factor.     

Constructing circular phylogenetic networks from weighted quartets using simulated annealing

In this paper, we present a heuristic algorithm based on the simulated annealing, SAQ-Net, as a method for constructing phylogenetic networks from weighted quartets. Similar to QNet algorithm, SAQ-Net constructs a collection of circular weighted splits of the taxa set. This collection is represented by a split network. In order to show that SAQ-Net performs better than QNet, we apply these algorithm to both the simulated and actual data sets containing salmonella, Bees, Primates and Rubber data sets. Then we draw phylogenetic networks corresponding to outputs of these algorithms using SplitsTree4 and compare the results. We find that SAQ-Net produces a better circular ordering and phylogenetic networks than QNet in most cases. SAQ-Net has been implemented in Matlab and is available for download at http://bioinf.cs.ipm.ac.ir/softwares/saq.net.     

Direct effects of triiodothyronine on production of anterior pituitary hormones and gonadal steroids in goldfish

The present study investigated the effects of triiodothyronine (T3) on pituitary gonadotropin (GTH) subunits, thyroid stimulating hormone (TSH) β subunit, and growth hormone (GH) mRNA levels, as well as gonadal steroid secretion during different stages of reproduction in goldfish. Goldfish pituitary cells cultured with T3 exhibited lower tshβ mRNA levels in all reproductive stages and lower luteinising hormone β (lhβ) mRNA levels in early recrudescence, whereas gh and fshβ mRNA levels were not altered. T3 injections significantly reduced circulating oestrogen (OE2) concentrations in early and mid recrudescent male goldfish, but were without effect on the circulating level of OE2 in female fish. T3 injections also reduced circulating levels of testosterone in both male and female goldfish during the mid stage of gonadal recrudescence. In vitro culture of goldfish ovarian follicles at the late stage of gonadal recrudescence, in the presence of T3, resulted in reduced OE2 secretion; no consistent effect of T3 on testosterone secretion was observed in cultured goldfish ovarian follicles and testis. These findings support the hypothesis that T3 impairs reproduction by inhibiting production of gonadal steroids and pituitary luteinising hormone production in goldfish. Mol. Reprod. Dev. 79: 592–602, 2012. © 2012 Wiley Periodicals, Inc.     

Calcium and other signalling pathways in neuroendocrine regulation of somatotroph functions

Relative to mammals, the neuroendocrine control of pituitary growth hormone (GH) secretion and synthesis in teleost fish involves numerous stimulatory and inhibitory regulators, many of which are delivered to the somatotrophs via direct innervation. Among teleosts, how multifactorial regulation of somatotroph functions are mediated at the level of post-receptor signalling is best characterized in goldfish. Supplemented with recent findings, this review focuses on the known intracellular signal transduction mechanisms mediating the ligand- and function-specific actions in multifactorial control of GH release and synthesis, as well as basal GH secretion, in goldfish somatotrophs. These include membrane voltage-sensitive ion channels, Na(+)/H(+) antiport, Ca(2+) signalling, multiple pharmacologically distinct intracellular Ca(2+) stores, cAMP/PKA, PKC, nitric oxide, cGMP, MEK/ERK and PI3K. Signalling pathways mediating the major neuroendocrine regulators of mammalian somatotrophs, as well as those in other major teleost study model systems are also briefly highlighted. Interestingly, unlike mammals, spontaneous action potential firings are not observed in goldfish somatotrophs in culture. Furthermore, three goldfish brain somatostatin forms directly affect pituitary GH secretion via ligand-specific actions on membrane ion channels and intracellular Ca(2+) levels, as well as exert isoform-specific action on basal and stimulated GH mRNA expression, suggesting the importance of somatostatins other than somatostatin-14.