Single Mutation in Shine-Dalgarno-Like Sequence Present in the Amino Terminal of Lactate Dehydrogenase of Plasmodium Effects the Production of an Eukaryotic Protein Expressed in a Prokaryotic System

One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5′-GGAGGC-3′ sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5′-GGAGGA-3′, by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.     

Nickel-induced changes in lipid peroxidation, antioxidative enzymes, and metal accumulation in Lemna gibba

In this study, an experiment was carried out to study the process of stress adaptation in Lemna gibba grown under nickel stress (0-20 mg Ni L(-1)). The results showed that Ni concentrations in plants increased with increasing Ni supply levels and reached a maximum of 142.82 mg.kg1 DW at 0.5 mg x L(-1) Ni treatments. The level of photosynthetic pigments (Chl a, Chi b, and total Chl) and soluble proteins increased upon exposure to high Ni concentrations. At the same time, the level of malondialdehyde (MDA) increased with increasing Ni concentration. These results suggested an alleviation of stress that was presumably the results of antioxidants such as superoxide dismutase (SOD), catalase (CAT) which generally increased linearly with increasing Ni levels. In addition, the proline content in L. gibba increased with increasing nickel levels. Our present work concluded that Lemna gibba has a high level of nickel tolerance and accumulation. We also found that moderate nickel treatment (0.05-5 mg x L(-1)) alleviated oxidative stress in plants, while the addition of higher amounts of nickel (10-20 mg x L(-1)) could cause an increasing generation of ROS, which was effectively scavenged by the antioxidative system. Therefore, L. gibba may be used as a phytoremediator in moderately polluted aquatic ecosystems.     

Comparative Analysis of the Protective Effects of Caffeic Acid Phenethyl Ester (CAPE) on Pulmonary Contusion Lung Oxidative Stress and Serum Copper and Zinc Levels in Experimental Rat Model

The aim of this study was to investigate the effects of caffeic acid phenethyl ester (CAPE) in the lungs by biochemical and histopathological analyses in an experimental isolated lung contusion model. Eighty-one male Sprague–Dawley rats were used. The animals were divided randomly into four groups: group 1 (n?=?9) was defined as without contusion and without CAPE injection. Group 2 (n?=?9) was defined as CAPE 10 μmol/kg injection without lung contusion. Group 3 (n?=?36) was defined as contusion without CAPE-administrated group which consisted of four subgroups that were created according to analysis between days?0, 1, 2, and 3. Group 4 (n?=?27) was defined as CAPE 10 μmol/kg administrated after contusion group divided into three subgroups according to analysis on days?1, 2, and 3. CAPE 10 μmol/kg was injected intraperitoneally 30 min after trauma and on days?1 and 2. Blood samples were obtained to measure catalase (CAT) and superoxide dismutase (SOD) activities and level of malondialdehyde (MDA) and for blood gas analysis. Trace elements such as zinc and copper were measured in serum. The lung tissue was also removed for histopathological examination. Isolated lung contusion increased serum and tissue SOD and CAT activities and MDA levels (p?<?0.05). Both serum and tissue SOD, MDA, and CAT levels on day?3 were lower in group 4 compared to group 3 (p?<?0.05). Further, the levels of SOD, MDA, and CAT in group 4 were similar compared to group 1 (p?>?0.05). CAPE also had a significant beneficial effect on blood gases (p?<?0.05). Both serum zinc and copper levels were (p?<?0.05) influenced by the administration of CAPE. Histopathological examination revealed lower scores in group 4 compared to group 3 (p?<?0.05) and no significant differences compared to group 1 (p?>?0.05). CAPE appears to be effective in protecting against severe oxidative stress and tissue damage caused by pulmonary contusion in an experimental setting. Therefore, we conclude that administration of CAPE may be used for a variety of conditions associated with pulmonary contusion. Clinical use of CAPE may have the advantage of prevention of pulmonary contusion.     

Potential of Raloxifene in reversing osteoarthritis-like alterations in rat chondrocytes: An in vitro model study

The aim of this study was to investigate the effects of Raloxifene (Ral) on degeneration-related changes in osteoarthritis (OA)-like chondrocytes using two- and three-dimensional models. Five-azacytidine (Aza-C) was used to induce OA-like alterations in rat articular chondrocytes and the model was verified at molecular and macrolevels. Chondrocytes were treated with Ral (1, 5 and 10 μM) for 10 days. Caspase-3 activity, gene expressions of aggrecan, collagen II, alkaline phosphatase (ALP), collagen X, matrix metalloproteinases (MMP-13, MMP-3 and MMP-2), and MMP-13, MMP-3 and MMP-2 protein expressions were studied in two-dimensional model. Matrix deposition and mechanical properties of agarose-chondrocyte discs were evaluated in three-dimensional model. One μM Ral reduced expression of OA-related genes, decreased apoptosis, and MMP-13 and MMP-3 protein expressions. It also increased aggrecan and collagen II gene expressions relative to untreated OA-like chondrocytes. In three-dimensional model, 1 μM Ral treatment resulted in increased collagen deposition and improved mechanical properties, although a significant increase for sGAG was not observed. In summation, 1 μM Ral improved matrix-related activities, whereas dose increment reversed these effects except ALP gene expression and sGAG deposition. These results provide evidence that low-dose Ral has the potential to cease or reduce the matrix degeneration in OA.     

Mutation of IGFBP7 causes upregulation of BRAF/MEK/ERK pathway and familial retinal arterial macroaneurysms

Insulin-like growth factor binding proteins (IGFBPs) play important physiological functions through the modulation of IGF signaling as well as IGF-independent mechanisms. Despite the established role of IGFs in development, a similar role for the seven known IGFBPs has not been established in humans. Here, we show that an autosomal-recessive syndrome that consists of progressive retinal arterial macroaneurysms and supravalvular pulmonic stenosis is caused by mutation of IGFBP7. Consistent with the recently established inhibitory role of IGFBP7 on BRAF signaling, the BRAF/MEK/ERK pathway is upregulated in these patients, which may explain why the cardiac phenotype overlaps with other disorders characterized by germline mutations in this pathway. The retinal phenotype appears to be mediated by a role in vascular endothelium, where IGFBP7 is highly expressed.     

Reactive Glia in the Injured Brain Acquire Stem Cell Properties in Response to Sonic Hedgehog

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Highlights? Stab wound injury and MCAo elicit a profound stem cell response ? Noninvasive brain injury fails to elicit a stem cell response ? SHH is upregulated and required in lesion conditions with a stem cell response ? SHH transducer deletion in astrocytes reduces their proliferative response to injury     

Targeting CD28, CTLA-4 and PD-L1 Costimulation Differentially Controls Immune Synapses and Function of Human Regulatory and Conventional T-Cells

CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.     

Covalent immobilization of catalase onto spacer-arm attached modified florisil: characterization and application to batch and plug-flow type reactor systems

Catalase was covalently immobilized onto florisil via glutaraldehyde (GA) and glutaraldehyde+6-amino hexanoic acid (6-AHA) (as a spacer arm). Immobilizations of catalase onto modified supports were optimized to improve the efficiency of the overall immobilization procedures. The V(max) values of catalase immobilized via glutaraldehyde (CIG) and catalase immobilized via glutaraldehyde+6-amino hexanoic acid (CIG-6-AHA) were about 0.6 and 3.4% of free catalase, respectively. The usage of 6-AHA as a spacer arm caused about 40 folds increase in catalytic efficiency of CIG-6-AHA (8.3 × 10? M?1 s?1) as compared to that of CIG (2.1 × 10? M?1 s?1). CIG and CIG-6-AHA retained 67 and 35% of their initial activities at 5 °C and 71 and 18% of their initial activities, respectively at room temperature at the end of 6 days. Operational stabilities of CIG and CIG-6-AHA were investigated in batch and plug-flow type reactors. The highest total amount of decomposed hydrogen peroxide (TAD-H?O?) was determined as 219.5 μmol for CIG-6-AHA in plug-flow type reactor.     

Evaluation of Antifungal Susceptibility Testing with Microdilution and Etest Methods of <Emphasis Type="Italic">Candida</Emphasis> Blood Isolates

Candida species that show an increasing number of clinical and/or microbiological resistance to several antifungals and are the most common agents of invasive fungal infections. The aim of this study was to investigate the in vitro susceptibility of Candida blood isolates to antifungal agents (amphotericin B, fluconazole, itraconazole, and voriconazole) by comparative use of the CLSI reference microdilution method and Etest. Four hundred Candida blood isolates (215 Candida albicans, 185 non-albicans Candida strains) were included in the study. The broth microdilution test was performed according to the CLSI M27 A2 document. Etest was carried out according to the manufacturer’s instructions. The MIC results obtained with reference microdilution were compared with those obtained with the Etest by using percent and categorical agreements. According to MIK₉₀ values, voriconazole was the most active and itraconazole was the least active drug in vitro against all Candida species. Other than voriconazole, statistically significant differences were found when the susceptibility of Candida albicans and non-albicans Candida spp. to amphotericin B, fluconazole, and itraconazole were compared. These antifungal agents were found to be more active to C. albicans. Among the non-albicans Candida species, the lowest MIC values were obtained for Candida parapsilosis isolates. When the standard method was compared with Etest, the total agreement was higher for C. albicans than for non-albicans species, especially for fluconazole and voriconazole. In view of the findings, it was concluded that itraconazole showed the lowest activity against all Candida species. Etest could be an alternative method in assessing the in vitro antifungal susceptibility of Candida spp., but it is more convenient to use the microdilution method for studying in vitro susceptibility of non-albicans species, in particular for those possessing high MIC values against azoles.