Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.     

Gene cloning and expression of a detergent stable alkaline protease from Aspergillus clavatus ES1

The genes, cDNA alpES1 and alpES1, encoding Aspergillus clavatus ES1 alkaline protease were amplified from complementary DNA (cDNA) and genomic DNA, respectively, cloned in pCR^®II-TOPO plasmid and then sequenced. Sequence analysis of the cDNA alpES1 gene revealed an open reading frame (ORF) of 1212 bp encoding a pre–pro-protein of 403 amino acid residues consisting of a 21-aa signal peptide, a 100-aa pro-peptide and a 282-aa mature protein with a calculated molecular weight of 28.5 kDa. Compared to the cDNA alpES1 gene, the alpES1 gene contained three introns, which had 53, 57 and 54 bp, respectively. The cDNA alpES1 gene was then sub-cloned in pET-30b(+) and expressed in Escherichia coli BL21 (λDE3). The purified recombinant protease had a molecular weight of about 32 kDa estimated by SDS-PAGE. Kinetic parameters, K_m and k_cat values of the recombinant AlpES1 for casein, were 0.23 mM and 12.38 min⁻¹, respectively. The catalytic efficiency (k_cat/K_m) was 53.82 min⁻¹ mM⁻¹.     

Protective effect of cannabidiol against cadmium hepatotoxicity in rats

The protective effect of cannabidiol, the non-psychoactive component of Cannabis sativa, against liver toxicity induced by a single dose of cadmium chloride (6.5 mg kg^?1 i.p.) was investigated in rats. Cannabidiol treatment (5 mg kg^?1/day, i.p.) was applied for five days starting three days before cadmium administration. Cannabidiol significantly reduced serum alanine aminotransferase, and suppressed hepatic lipid peroxidation, prevented the depletion of reduced glutathione and nitric oxide, and catalase activity, and attenuated the elevation of cadmium level in the liver tissue resulted from cadmium administration. Histopathological examination showed that cadmium-induced liver tissue injury was ameliorated by cannabidiol treatment. Immunohistochemical analysis revealed that cannabidiol significantly decreased the cadmium-induced expression of tumor necrosis factor-α, cyclooxygenase-2, nuclear factor-κB, caspase-3, and caspase-9, and increased the expression of endothelial nitric oxide synthase in liver tissue. It was concluded that cannabidiol may represent a potential option to protect the liver tissue from the detrimental effects of cadmium toxicity.     

Synthesis and evaluation of modified chalcone based p53 stabilizing agents

Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (CAL-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI₅₀ values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI₅₀ of 0.473 ± 0.043 µM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-h post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities.     

Protective effect of combined therapy with dithiothreitol,zinc and selenium protects acute mercury induced oxidative injury in rats

The present study was undertaken to establish mode of action, comparative therapeutic efficacy and safety evaluation of dithiothreitol (DTT) supplemented with Zn and Se against dimethylmercury in rats. Adult male albino rats of Sprague-Dawley strain (150 ± 10 g, n = 6 per group) were exposed a bolus dose of dimethylmercury (10 mg/kg, p.o.) for once only followed by DTT (15.4 mg/kg, i.p.) along with the combination of antioxidants Zn and Se (2 mmol/kg and 0.5 mg/kg, p.o.) after 72 h of toxicant administration for three days. The results showed a significant (P ≤ 0.05) increase in the activities of AST, ALT, alkaline phosphatase, lactate dehydrogenase, in serum after toxicant administration. This was accompanied by histopathological observations. A significant rise was observed in lipid peroxidation level and mercury ion concentration however reduced glutathione content decreased in liver, kidney and brain. A significant (P ≤ 0.05) decrease in the activity of acetyl cholinesterase was also seen in different regions of brain. Combined treatment of DTT along with Zn and Se significantly (P ≤ 0.05) recouped the alterations in the enzymatic activities of serum and reversed the tissue biochemical and histopathological changes of liver, kidney and brain. Our results demonstrate that combined treatment of thiol chelator (DTT) along with antioxidants (Zn and Se) plays an important role against dimethylmercury induced tissue damage and hepatic, nephro and neurotoxicity.     

Construction and characterization of a cDNA library from shark regenerated hepatic tissue

Sharks are a type of fish with a full cartilaginous skeleton and have big livers. To better understand liver regeneration in sharks and screening for the important genes participated in disease-defense, in this study, a cDNA library of regenerated liver tissues of shark, Chiloscyllium plagiosum, was constructed. A total of 2103 expressed sequence tags (ESTs), which represents 997 unique genes, were sequenced. Among these genes, 434 (43.53%) of them showed significant similarity (E-values < 10^?5) to the sequences in NCBI Nt database, 685 (68.71%) of these unique genes showed significant similarity (E-values < 10^?5) to the sequences in NCBI Nr database, and 662 (66.40%) of these unique genes showed significant similarity (E-values < 10^?5) to the Swiss-Prot database. Preliminary analysis of unique genes according to COG database showed that unigenes were further grouped into 21 functional categories including inorganic ion transport and metabolism, energy production and conversion, posttranslational modification, protein turnover and chaperones, general function prediction only, translation, and ribosomal structure and biogenesis. Several possible candidate genes involved in liver regeneration were selected to analyze their expression with relative quantification real-time PCR. This study may contribute to our better understanding of the molecular mechanism of regeneration in shark liver. Furthermore, the EST cataloguing and profiling of shark will be also benefited to the functional genomic research in this marine species.     

Clearance of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) and immunological changes in experimentally injected Macrobrachium rosenbergii

Macrobrachium rosenbergii was experimentally challenged with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) to study the clearance of these viruses and consequent changes in various immunological parameters. The healthy animals were injected MrNV and XSV intramuscularly and various organ samples such as gill tissue, head soft tissue, pleopods and intestine were collected at different time intervals of 3, 5, 10, 15, 25, 50, 75 and 100 d post-infection (p.i.) to study the viral clearance. Tissue tropism and clearing of MrNV and XSV were confirmed by RT-PCR, nested RT-PCR and bioassay. These 2 viruses failed to cause mortality or clinical signs of disease in injected adult prawns during the experimental period of 100 days. The result of RT-PCR analysis revealed that all the organs showed positive for both viruses by single step RT-PCR on 3, 5 and 10 d p.i., positive by nested RT-PCR on 15 and 20 d p.i. and all the organs became negative at 25 d p.i. onwards. The viral inoculum prepared from the tissue of MrNV and XSV-injected M. rosenbergii at 3, 5, 10, 15 and 20 d p.i. caused 100% mortality in post-larvae of M. rosenbergii at 9, 8, 7, 10 and 10 d p.i., respectively whereas the inoculum prepared at 25, 50 and 100 d p.i. failed to cause significant mortality in post-larvae of prawn. Immunological parameters such as proPO, superoxide anion, SOD, THC, clotting time and oxyhemocyanin were determined in MrNV and XSV-injected prawns and significant differences in some of the immunological parameters were found in the early days p.i. and became insignificant in the later days p.i.     

Discovery of novel 4(1H)-quinolone derivatives as potential antiproliferative and apoptosis inducing agents

A series of novel 4(1H)-quinolone derivatives was synthesized and evaluated for antiproliferative activity in vitro. The results showed that these compounds exhibited more potent antiproliferative effect against a panel of human tumor cell lines than the lead compound 7-chloro-4(1H)-quinolone 1. Compound 7e was found to be the most potent antiproliferative agent and to exhibit selective cytotoxic activity against HepG2 cell lines with IC₅₀ value lower than 1.0 μM. Annexin V/FITC-PI assay showed that compound 7e induced apoptosis in HepG2 cells with a dose-dependent manner. Western blotting analysis indicated that compound 7e induced cell cycle arrest in G2/M phase by p53-depedent pathway.     

Phenylethanoid glycosides from the leaves of Magnolia hodgsonii

Three new phenylethanoid glycosides, 2-(3-hydroxy-4-methoxyphenyl)ethyl 1-O-β-d-allopyranoside (hodgsonialloside A, 1), 2-(3-hydroxy-4-methoxyphenyl)ethyl 1-O-β-d-glucopyranosyl-(1 → 4)-β-d-allopyranoside (hodgsonialloside B, 2) and 2-(3-methoxy-4-hydroxyphenyl)ethyl 1-O-β-d-allopyranoside (hodgsonialloside C, 3) were isolated from the leaves of Magnolia hodgsonii in addition to six known compounds, tyrosol 4-O-β-d-xylopyranosyl-(1 → 6)-β-d-glucopyranoside (4), kaempferol 3-O-neohesperidoside (5), kaempferol 3-O-rutinoside (6), kaempferol 3-O-α-l-rhamnopyranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside (7), (+)-syringaresinol O-β-d-glucopyranoside (8), and oblongionoside C (9). The structure elucidation of these compounds was based on analyses of physical and spectroscopic data including 1D and 2D NMR experiments.     

‘3 + 1’ mixed-ligand oxorhenium(V) complexes and their inhibition of the cysteine proteases cathepsin B and cathepsin K

The synthesis of several new oxorhenium(V) complexes containing the ‘3 + 1’ mixed-ligand donor set, ReO(SXS)(SR) (where X = S, O, N(R′); R = alkyl, aryl, heterocylce; R′ = H, alkyl, aryl), is described. The X-ray structure for four of these complexes ReO(SN(Ph)S)(SPh) (6), ReO(SN(CH₂CH₂NMe₂)S)(SPhOMe-p) (10), ReO(SOS)(SPh) (29) and ReO(SOS)(SPhNO₂-p) (30) was determined. The inhibitory activity of all of the oxorhenium(V) complexes reported herein was evaluated against the cysteine proteases cathepsin B and K in vitro. Compound 25, ReO(SSS)(S-4py) · HCl, was the best inhibitor of the series against cathepsin B with an IC₅₀ of 10 nM. Several of the complexes exhibited specificity for cathepsin B over K, suggesting that oxorhenium(V) complexes can be designed to be enzyme specific. The results described in this paper show that the oxorhenium(V) ‘3 + 1’ complexes are potent inhibitors of cathepsin B and K, constituting promising potential for the treatment of cancer and osteoporosis, respectively.     

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2 g/L of AECC and produce 22.1 g/L of solvents (4.25 g/L acetone, 11.5 g/L butanol and 6.37 g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.