Expression and Functional Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Recombinant <Emphasis Type="SmallCaps">l</Emphasis>.Asparaginase

Recombinant _l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg²⁺ and inhibited by Zn²⁺ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as _L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.     

The dsRNA-mimetic poly (I:C) and IL-18 synergize for IFNγ and TNFα expression

Interleukin (IL)-18 bioactivity and dsRNA sensing by receptors of innate immunity are key components of anti-viral host defense. Despite extensive data on signal transduction activated by both pathways knowledge on cross-communication is incomplete. By using human PBMC and predendritic KG1 cells, as prototypic IL-18-responsive cellular models, we sought to assess cytokine production under the influence of IL-18 and the dsRNA-mimetic poly (I:C). Here, we report on potent synergy between both mediators concerning pro-inflammatory IFNγ and TNFα production. KG1 data revealed that synergistic induction likely relied on TLR3 and was associated with prolonged/increased activation of NF-κB, as detected by IκB analysis and luciferase reporter assays, respectively. Moreover, extended activation of JNK was mediated by IL-18/poly (I:C). Although vital for innate immunity, overwhelming induction of inflammatory cytokines during viral infections poses the threat of serious collateral tissue damage. The stunning synergism inherent to IL-18/dsRNA-induced TNFα/IFNγ detected herein may contribute to this pathological phenomenon.     

Significant curative functions of the mesenchymal stem cells on methotrexate‐induced kidney and liver injuries in rats

Mesenchymal stem cells (MSCs) curative effects on methotrexate (MTX)‐induced kidney and liver injuries remain elusive. Therefore, rats were divided into five groups, rats received MTX orally (14 mg/kg) as a single dose/week for 2 weeks, groups 3 and 4 were injected once with 2 × 10⁶ cells bone marrow MSCs and adipose‐derived MSCs, respectively. The last group administered dexamethasone (DEX) (0.5 mg/kg, p.o) for 7 days. MTX caused marked increase in malondialdehyde and nitrite/nitrate concentrations. However, MTX administration decreased reduced glutathione content plus catalase activity. In addition, MTX caused a significant increment in kidney and liver biomarkers levels. Moreover, MTX showed renal tubules vacuolation and necrosis of hepatocytes, as well expression of caspase‐3 and nuclear factor kappa beta in kidney and liver tissues were observed. MSCs treatment alleviated previous side effects induced by MTX. MSCs improved nephrotoxicity and hepatotoxicity induced by MTX to a better extent as compared with DEX.     

Curcumin ameliorates diclofenac sodium‐induced nephrotoxicity in male albino rats

Exposure to drugs often results in toxicity in the kidney which represents the major control system maintaining homeostasis of the body and thus is especially susceptible to xenobiotics. Nephrotoxicity is a life‐threatening side‐effect of nonsteroidal anti‐inflammatory drugs (NSAIDs). Diclofenac is one of the most frequently prescribed NSAIDs and have been reported to cause multiple organs damage. Curcumin (CUR) exhibits nephroprotective properties. Therefore, rats were divided into four groups; rats of groups 3 and 4 received diclofenac (100 mg/kg, i.m.), whereas rats of groups 2 and 4 received CUR (100 mg/kg, p.o.) for 3 days. Diclofenac revealed a significant increase in urea and creatinine levels and malondialdehyde concentration and marked reduction in catalase activity and reduced glutathione concentration. Histopathologically, diclofenac produced fatty changes and eosinophilic casts were detected in the renal tubules, those were attenuated by administration of CUR prior diclofenac.     

Assessment of anti-mutagenic,anti-histopathologic and antioxidant capacities of Egyptian bee pollen and propolis extracts

Bee pollen and propolis are popular, traditional health foods. The objective of the current study was to investigate the anti-mutagenic, anti-histopathologic and antioxidant effects among water extracts of Egyptian bee pollen (WEBP) and brown powder of water-soluble derivative propolis (WSDP) on cisplatin (CDDP) induced hepatic, renal, testicular and genotoxicity in male albino mice (Mus muscullus), in addition to their effects on the oxidant/antioxidant status in the tested organs. Hepatic, renal and testicular dysfunctions were evaluated histologically; while genotoxicity and cytotoxicity were evaluated by the bone marrow chromosomal aberration assay and mitotic index, respectively. Moreover, oxidative stress was explored via determination of lipid peroxidation, catalase activity and the concentration of the reduced form of glutathione. The treatment of mice with WEBP and WSDP at doses 140 and 8.4 mg/kg b. wt./day, respectively for 14 days simultaneously with CDDP (2.8 mg/kg b. wt.) resulted in significant protection. The positive control animals taken CDDP alone showed toxic histological and genetical manifestations (at P < 0.05) accompanied with an elevated content of peroxidized lipid and lowered catalase activity and glutathione concentration in the homogenate of liver, kidney and testis tissues (at P < 0.001). These toxic side effects in all tested organs were greatly ablated with a significant reduction in lipid peroxidation level and elevation in catalase activity and glutathione concentration (P < 0.001) when using both WEBP and WSDP. On the basis of the present assays, Bee pollen appears more potent in exerting an ameliorative effect and this effect was more pronounced in testis.     

Molecular diagnosis of Helicobacter pylori antibiotic resistance in the Taif region,Saudi Arabia

Success in eradication of Helicobacter pylori is declining globally because H. pylori has developed resistance against most of the antibiotics proposed for eradication regimens, mainly through point mutations. The present study included 200 patients with dyspepsia attending Taif Hospital. Gastric biopsies were obtained during gastroscopy and subjected to rapid urease testing. Molecular methods were used to confirm diagnoses of H. pylori infection and to identify resistance gene variants of four antibiotics; namely, clarithromycin, metronidazole, fluoroquinolones and tetracycline (23S rRNA, gyrA, rdxA and 16S rRNA respectively). Of all investigated patients, Molecular diagnoses were made in 143 of all investigated patients; thus, the prevalence was .5%. The overall rate of resistance to clarithromycin among the H. pylori‐positive patients was high (39.9%) and the rate of resistance significantly greater (48.2%) among the secondary resistance group, secondary resistance being defined as resistance as a result of previous exposure to the relevant antibiotic. The rate of resistance to fluoroquinolones was considered moderate; the difference in rate of resistance between the primary and secondary resistance groups (8.4% and 9.5%, respectively) was not significant Also, there was a low prevalence of both primary and the secondary tetracycline resistance in the study cohort. In contrast, the prevalence of metronidazole resistance was considered high with no significant difference between the two resistance groups. H. pylori showed an increased prevalence of resistance to all four of the commonly used therapeutic agents. Thus, eradication therapy should be based on the regional results of susceptibility testing. Moreover, treatment tailored according to individually determined H. pylori susceptibility may be a reasonable future goal.     

Chemical Composition of Aqueous Ethanol Extract of Luffa cylindrica Leaves and Its Effect on Representation of Caspase‐8, Caspase‐3, and the Proliferation Marker Ki67 in Intrinsic Molecular Subtypes of Breast Cancer in Vitro

Breast cancer constitutes the second most prevalent cancer in Egypt, the problem needs more trends in treatment and treatment development either by regimen modification or introducing new drugs, and the main objective of this study is to screen the effects of the aqueous ethanol herbal extract of Luffa cylindrica leaves on different types of breast cancer cell lines representing different molecular subtypes of the disease. The major active constituents of the extract were tentatively identified by LC/MS which revealed the presence of phenolic compound derivatives and saponin that may be responsible in part for the activity of the extract. The emphasis was laid on the main apoptotic pathways as well as the extract effect on the normal cell line. Results of phytochemical investigation, cell cycle analysis, and molecular analysis of apoptotic and proliferative markers have shown effective anticancer activity against MCF‐7, BT‐474, and MDA‐MB‐231 cell lines which represent three subtypes of breast cancer, luminal A, luminal B, and triple negative, respectively. On the other hand, the effects on normal lung fibroblast cell line are less prominent at the dose used for treating breast cancer cell lines.     

Iron-mediated retinal degeneration in haemojuvelin-knockout mice

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE (human leucocyte antigen-like protein involved in iron homoeostasis), transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Recent studies have established the expression of all of the five genes in the retina, indicating their importance in retinal iron homoeostasis. Previously, we demonstrated that HJV is expressed in RPE (retinal pigment epithelium), the outer and inner nuclear layers and the ganglion cell layer. In the present paper, we report on the consequences of Hjv deletion on the retina in mice. Hjv-/- mice at ≥18 months of age had increased iron accumulation in the retina with marked morphological damage compared with age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv-/- mice included hyperplasia of RPE. We isolated RPE cells from wild-type and Hjv-/- mice and examined their growth patterns. Hjv-/- RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv-/- RPE cells also showed up-regulation of Slc7a11 (solute carrier family 7 member 11 gene), which encodes the 'transporter proper' subunit xCT in the heterodimeric amino acid transporter xCT/4F2hc (cystine/glutamate exchanger). BMP6 (bone morphogenetic protein 6) could not induce hepcidin expression in Hjv-/- RPE cells, confirming that retinal cells require HJV for induction of hepcidin via BMP6 signalling. HJV is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated HJV is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these results confirm the biological importance of HJV in the regulation of iron homoeostasis in the retina and in RPE.