Mutant MMP-9 and HGF Gene Transfer Enhance Resolution of CCl4-Induced Liver Fibrosis in Rats: Role of ASH1 and EZH2 Methyltransferases Repression

Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl₄ induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl₄ for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. Conclusion: Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and EZH2 in resolution of liver fibrosis.     

Evaluating germinability of eight desert halophytes under long-term seed storage: Implications for conservation

Ex situ conservation in seed banks is a potential complementary conservation strategy for native plant species.It is well established that ex situ seed banking of native wild plants prolongs seed viability and thereby preserves genetic and species diversity for future use.We evaluated ex situ storage potential of eight halophytic species from deserts in the United Arab Emirates(UAE)by studying seed germination.Specifically,we examined the germinability of freshly collected seeds and seeds stored for three years in a seed bank.We also examined the effect of light conditions on fresh and stored seed germination.Fresh seeds of seven of the eight species tested had a higher germination rates under 12/12 h light/dark fluctuations than did those exposed to total darkness.Storage reduced light sensitivity in Halocnemum strobilaceum,Suaeda aegyptiaca,Salsola drummondii and Salsola imbricata,but increased the requirement for light in Arthrocnemum macrostachyum.In Anabasis setifera,storage decreased germination percentage when there was a 12-hour light/dark fluctuation,but increased germination rate when exposed to the dark treatment.Storage significantly reduced germination in both the light/dark and dark treatments in Suaeda vermiculata and S.aegyptiaca.Germination speed also responded differently to storage;whereas Timson's index significantly increased in A.macrostachyum and H.strobilaceum,it significantly decreased for S.drummondii,S.aegyptiaca and S.vermiculata.Germination of these species at a range of temperatures requires further testing;additionally,we strongly suggest that these laboratory findings be complemented by field studies.     

Understanding the role of the trifluoromethyl group in reactivity and regioselectivity in [3+2] cycloaddition reactions of enol acetates with nitrones. A DFT study

The mechanism of the [3+2] cycloaddition (32CA) reaction of C-phenyl-N-methylnitrone with ethyl trifluoroacetoacetate has been theoretically studied at the MPWB1K/6-311G(d,p) level. This 32CA reaction, in which the enol form of the β-keto ester participates as the ethylene component, takes place with complete ortho regioselectivity and exo stereoselectivity. The presence of the CF₃ group in the β-position in the enol acetate accelerates the 32CA reaction, but it does not modify the regioselectivity, which is controlled by the presence of the ester group. While ortho regioselectivity is reproduced by the MPWB1K calculations, the endo selectivity is not. The inclusion of solvent effects slightly decreases the reactivity but does not modify the gas phase selectivities. Analysis of the DFT global reactivity indices and the Parr functions in reagents provide a rationalization for the participation of ethyl trifluoroacetoacetate and the regioselectivity in this zw-type 32CA reaction.     

Synthesis of new 2-substituted 6-bromo-3-methylthiazolo[3,2-alpha]-benzimidazole derivatives and their biological activities

1-(6-Bromo-3-methyl-1,3-thiazolo[3,2-alpha]benzimidazol-2-yl)ethanone (2) was prepared by bromination at ambient temperature of 1-(3-methylthiazolo[3,2-alpha]benzimidazol-2-yl)ethanone (1). The structure of 2 was determined by single-crystal X-ray diffraction. The precursor 5 was synthesized by heating a mixture of acetyl 2 and bromine. Various 2-substituted 6-bromo-3-methylthiazolo[3,2-alpha]benzimidazoles containing 1,3-thiazole, 1,4-benzothiazine, quinoxaline or imidazo[1,2-alpha]pyridine moieties were prepared starting from bromoacetyl 5. Taken together from the biological investigations, 2, 5, and 7a were potent immunosuppressors against both macrophages and T-lymphocytes, and 7b, 11b, and 14 were potent immunostimulators towards both types of immune cells. The results also revealed that, among others, 2 and 14 were the most significant inhibitors of LPS-stimulated NO generation, and that 5, 7a, and 7b had a weak radical scavenging activity against DPPH radicals. Moreover, 2, 5, and 7a had a concomitant strong cytotoxicity against colon carcinoma, hepatocellular carcinoma, and lymphoblastic leukemia cells. Collectively, compounds 2, 5, and 7a are multipotent compounds with promising biological activities.     

Erythrocyte Hemolysis and Hemoglobin Oxidation Promote Ferric Chloride-induced Vascular Injury

The release of redox-active iron and heme into the blood-stream is toxic to the vasculature, contributing to the development of vascular diseases. How iron induces endothelial injury remains ill defined. To investigate this, we developed a novel ex vivo perfusion chamber that enables direct analysis of the effects of FeCl₃ on the vasculature. We demonstrate that FeCl₃ treatment of isolated mouse aorta, perfused with whole blood, was associated with endothelial denudation, collagen exposure, and occlusive thrombus formation. Strikingly exposing vessels to FeCl₃ alone, in the absence of perfused blood, was associated with only minor vascular injury. Whole blood fractionation studies revealed that FeCl₃-induced vascular injury was red blood cell (erythrocyte)-dependent, requiring erythrocyte hemolysis and hemoglobin oxidation for endothelial denudation. Overall these studies define a unique mechanism of Fe³⁺-induced vascular injury that has implications for the understanding of FeCl₃-dependent models of thrombosis and vascular dysfunction associated with severe intravascular hemolysis.Iron and heme-containing moieties are indispensable for the normal transport of oxygen in the blood; however, once released into the bloodstream these molecules are highly toxic to the vasculature because of their pro-oxidative effects on the endothelium (1-3). Humans have therefore evolved sophisticated iron transport and sequestration systems as well as heme-metabolizing enzymes to rapidly clear iron and heme from the circulation (4, 5). There is growing evidence that defects in these natural protective mechanisms lead to endothelial dysfunction and vascular disease, and as a consequence, methods that reduce the pro-oxidative effects of iron and heme may have therapeutic benefit (2).Clinical syndromes associated with marked intravascular hemolysis and circulating free hemoglobin, such as sickle cell disease, paroxysmal nocturnal hemoglobinuria, thalassemias, and hereditary spherocytosis, lead to endothelial dysfunction, thrombosis, and vascular disease (5-10). Similarly administration of purified recombinant hemoglobin to humans promotes vascular injury and arterial thrombosis, precipitating acute myocardial infarction (11-13). Some of these vascular effects are related to nitric oxide scavenging by excess plasma hemoglobin, whereas others are linked to cytotoxic, proinflammatory, and pro-oxidant effects of iron-containing hemoglobin and heme (14-19). Interestingly elevated levels of body iron stores are associated with an increased risk of myocardial infarction, and carriers of the hemochromatosis gene have an increased risk of myocardial infarction and cardiovascular death (20, 21). Whether the pro-oxidative effects of iron per se are proatherogenic remains controversial; however, in the context of erythrocyte-dependent release of hemoglobin and heme, redox-active iron is likely to play an important role in promoting vascular dysfunction.The well defined pro-oxidative properties of redox-active iron have been exploited experimentally with topical application of ferric chloride (FeCl₃) widely used to induce vascular injury and thrombosis in experimental animal models (22). High concentrations of FeCl₃ induce profound injury to the vasculature, leading to endothelial denudation, and collagen and tissue factor exposure, leading to the rapid formation of vaso-occlusive thrombi. Histologically FeCl₃-induced thrombi are rich in platelets, fibrin, and red blood cells (23-26). However, the mechanism(s) by which FeCl₃ induces vascular injury has not been clearly defined. FeCl₃ can have direct pro-oxidative effects on endothelial cells as a result of the Fenton reaction, leading to hydroxyl radical generation and lipid peroxidation (1, 3). It can also mediate vascular injury indirectly through oxidative modification of LDL³ (3, 14). A recent study has demonstrated transfer of ferric ions through the vasculature, penetrating the internal elastic membrane and emerging through the endothelium via an endocytic/exocytic pathway, leading to the development of ferric oxide aggregates in the vascular lumen (27). Although the direct cytotoxic effects of redox-active iron on endothelial cells have been well established in vitro, the importance of this mechanism to the severe vascular injury and thrombus formation induced by topical FeCl₃ in vivo remains unclear.To gain insight into this, we developed a novel ex vivo perfusion chamber that enables direct analysis of the effects of FeCl₃ on the vasculature. Our studies demonstrated that FeCl₃ alone induces relatively mild injury to endothelial cells with severe vascular injury only observed in the presence of flowing blood. Whole blood fractionation studies revealed that FeCl₃-mediated vascular injury is dependent on erythrocyte hemolysis and hemoglobin oxidation, defining a unique mechanism of iron-induced vascular injury.     

Direct detection of unamplified spring viraemia of carp virus RNA using unmodified gold nanoparticles

Spring viraemia of carp (SVC) is a viral disease that mainly affects carp Cyprinus carpio and other cyprinid fish, causing severe economic losses. Rapid detection and identification of spring viraemia of carp virus (SVCV) is crucial for effective disease management. Recent advances in nanoscience are having a significant impact on many scientific fields, especially biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. Single- and double-stranded oligonucleotides can be adsorbed on gold nanoparticles (AuNPs) in colloidal solution under certain conditions. We exploited this phenomenon to develop a specific hybridization assay for direct detection of SVCV-RNA without prior amplification. The result of the hybridization process could be detected visually within 1 min when the colour of the reaction mixture changed from red to blue (positive reaction) or remains red (negative). The lower detection limit of the assay was estimated to be 10-3 TCID50 ml-1 SVCV-RNA, and it has the feasibility to detect the target virus-RNA in clinical specimens without previous amplification. In order to obtain an indication of the assay's performance on clinical samples we compared the optimized assay with nested RT-PCR in detection of SVCV-RNA in infected fish samples. The concordance of the 2 methods was defined as 100% when compared to nested RT-PCR positive and negative samples. The SVC-AuNPs assay requires only 15 min, eliminates the need for thermal cycling or detection instruments and is a specific and rapid tool for detection of SVCV-RNA directly from clinical samples.     

Effects of hydrogen cyanamide on antioxidant enzymes’ activity,proline and polyamine contents during bud dormancy release in Superior Seedless grapevine buds

The effect of hydrogen cyanamide (HC) on dormancy release, antioxidant enzyme’s activity and proline and free polyamine contents were investigated in ‘Superior Seedless’ grapevine buds. HC application caused a sharp decrease of catalase (CAT, EC 1.11.1.6) activity and a transient stimulation during the 5 days following treatment of peroxidase (POD, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11) activities. This coincided with an accumulation of total free polyamines, especially putrescine (Put). Proline content increased dramatically. There was a strong correlation between APX and POD activities and total free PAs and Put contents implying a possible stimulating effect of the latter compounds on these enzymes. These observations indicate that HC triggers an oxidative stress leading to bud endodormancy release. Afterward, as budbreak started, we observed a rapid proline and Put degradation; this could be responsible for reactivation of growth. Indeed, the decline in Put to (Spd + Spm) ratio, reported here, may be considered as a reliable biochemical marker of bud growth resumption.     

Pharmacokinetics, immunogenicity and anticancer efficiency of Aspergillus flavipesl-methioninase

Methionine starvation can powerfully modulate DNA methylation, cell cycle transition, polyamines and antioxidant synthesis of tumor cells, in contrary to normal ones. Aspergillus flavipesl-methioninase was previously characterized by our studies, displaying affordable biochemical properties comparing to Pseudomonas putida enzyme (ONCASE). Thus, the objective of current study was to evaluate the catalytic properties of Af-METase in New Zealand rabbits, exploring its antitumor efficacy. In vivo, Af-METase (40.8U/ml) have T(1/2) 19.8h, elimination constant 0.088U/h and apparent volume distribution 85U/ml. Also, Af-METase has two maxima one at A(280nm) (apo-enzyme) and at A(420nm) (internal Schiff base of PLP), unlike control plasma (without enzyme). The two peaks of absorption spectra were detected maximally at 15min then the absorbance at 420nm was subsequently decreased with circulation time, due to dissociation of the co-enzyme. The A(280/420) ratio was increased from 1.69 to 5.81 with circulation time from 15 to 30h. Rabbits plasma methionine was depleted from 18.7μM (control) to 8.8μM after 1h of enzyme injection and completely omitted after 2h till 19h, assuming the sustainability of negligible levels of methionine (<2μM) in plasma of rabbits, for about 17h. Upon infusion of PLP, the T(1/2) of Af-METase was significantly prolonged by 3.2 fold, assuming the fully reconstitution of the enzyme. The holo-AfMETase still retained its co-enzyme, completely, till 33h of PLP infusion. From spectral studies, the internal aldimine linkage of apo-Af-METase was constructed upon PLP infusion, with fully catalytic structure after less than 4h of its infusion, the A(280/420) ratio being not relatively changed till 45h. After 25 days of last enzyme dose, the titer of IgG was increase by about 1.66 fold comparing to control (without enzyme). However, IgM was not detected along the tested challenge points. In vitro, plasma anti-Af-METase neutralizing antibodies (NAb) were assessed, with no significant reduction on activity of Af-METase by Nab. All the hematological parameters were in normal range, otherwise, the RBCs titer and platelet level was slightly increased, after 25 days of Af-METase injection, comparing to control. There is no obvious negative effect on chemistry of liver, kidney, glucose, lipids, and other electrolytes. Additionally, the anticancer activity of Af-METase was evaluated against five types of human cancer cell lines, in vitro. The enzyme showed a powerful activity against prostate (PC3), liver (HEPG2) and breast (MCF7) cancers, with IC(50) 0.001U/ml, 0.26U/ml and 0.37U/ml, respectively.     

Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.