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.     

Epidemiological Characteristics of Strongyloidiasis in Inhabitants of Indigenous Communities in Borneo Island,Malaysia

Epidemiological study on strongyloidiasis in humans is currently lacking in Malaysia. Thus, a cross-sectional study was carried out to determine the prevalence of Strongyloides stercoralis infection among the inhabitants of longhouse indigenous communities in Sarawak. A single stool and blood sample were collected from each participant and subjected to microscopy, serological and molecular techniques. Five species of intestinal parasites were identified by stool microscopy. None of the stool samples were positive for S. stercoralis. However, 11% of 236 serum samples were seropositive for strongyloidiasis. Further confirmation using molecular technique on stool samples of the seropositive individuals successfully amplified 5 samples, suggesting current active infections. The prevalence was significantly higher in adult males and tended to increase with age. S. stercoralis should no longer be neglected in any intestinal parasitic survey. Combination of more than 1 diagnostic technique is necessary to increase the likelihood of estimating the ‘true’ prevalence of S. stercoralis.     

Impact of dengue virus (serotype DENV-2) infection on liver of BALB/c mice: A histopathological analysis

In this research, we characterized the histopathological impact of dengue virus (serotype DENV-2) infection in livers of BALB/c mice. The mice were infected with different doses of DENV-2 via intraperitoneal injection and liver tissues were processed for histological analyses and variation was documented. In the BALB/c mouse model, typical liver tissues showed regular hepatocyte architecture, with normal endothelial cells surrounding sinusoid capillary. Based on histopathological observations, the liver sections of BALB/c mice infected by DENV-2 exhibited a loss of cell integrity, with a widening of the sinusoidal spaces. There were marked increases in the infiltration of mononuclear cells. The areas of hemorrhage and micro- and macrovesicular steatosis were noted. Necrosis and apoptosis were abundantly present. The hallmark of viral infection, i.e., cytopathic effects, included intracellular edema and vacuole formation, cumulatively led to sinusoidal and lobular collapse in the liver. The histopathological studies on autopsy specimens of fatal human DENV cases are important to shed light on tissue damage for preventive and treatment modalities, in order to manage future DENV infections. In this framework, the method present here on BALB/c mouse model may be used to study not only the effects of infections by other DENV serotypes, but also to investigate the effects of novel drugs, such as recently developed nano-formulations, and the relative recovery ability with intact immune functions of host.     

Chemical Composition,Antioxidant Properties, α‐Glucosidase Inhibitory,and Antimicrobial Activity of Essential Oils from Acacia mollissima and Acacia cyclops Cultivated in Tunisia

The genus Acacia is quite large and can be found in the warm subarid and arid parts, but little is known about its chemistry, especially the volatile parts. The volatile oils from fresh flowers of A. mollissima and A. cyclops (growing in Tunisia) obtained by hydrodistillation were analyzed by GC then GC/MS. Eighteen (94.7% of the total oil composition) and 23 (97.4%) compounds were identified in these oils, respectively. (E,E)‐α‐Farnesene (51.5%) and (E)‐cinnamyl alcohol (10.7%) constituted the major compounds of the flower oil of A. mollissima, while nonadecane (29.6%) and caryophyllene oxide (15.9%) were the main constituents of the essential oil of A. cyclops. Antioxidant activity of the isolated oils was studied by varied assays, i.e., 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2‐azinobis 3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS); the isolated oils showed lowest IC₅₀ (4 – 39 μg/ml) indicating their high antioxidant activity. The α‐glucosidase inhibitor activity was also evaluated and Acacia oils were found to be able to strongly inhibit this enzyme with IC₅₀ values (81 – 89 μg/ml) very close to that of acarbose which was used as positive control. Furthermore, they were tested against five Gram‐positive and Gram‐negative bacteria and one Candida species. Essential oil of A. mollissima was found to be more active than that of A. cyclops, especially against Pseudomonas aeruginosa (MIC = 0.31 mg/ml and MBC = 0.62 mg/ml).     

In vitro spermicidal activity of peptides from amphibian skin: dermaseptin S4 and derivatives

Sexually transmitted infections and unplanned pregnancies present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention and contraception are needed urgently. In the present study, efforts were made to evaluate the contraceptive potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and its derivatives, a set of chemically modified peptides was synthesized and evaluated. Normal human semen samples were used to detect the spermicidal activity of the new compounds. HeLa cultures were used to determine the safety of compounds toward their toxicity. Fluorescent-binding assays were performed to evaluate the rapidity and the irreversibility of the sperm-immobilizing activity of peptides. All DS4 derivatives elicited concentration-dependent spermicidal activity at microgram concentrations (EC(100) values: 25 microg/ml-l mg/ml). The order was K4S4=S4a>S4>K4S4(1-16)a>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than nonoxynol-9, the most widely used spermicide, and their activity was not accompanied by total loss of plasma membrane integrity as detected by fluorescent microscopy. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the spermicidal effect. This study indicates that dermaseptins are spermicidal molecules that deserve to be tested as topical contraceptive with useful activities that can add to their prophylaxis, safety, and effectiveness.     

Contribution of extended-spectrum AmpC (ESAC) beta-lactamases to carbapenem resistance in Escherichia coli

ESAC beta-lactamases have increased catalytic efficiencies toward extended-spectrum cephalosporins and to a lesser extent toward imipenem as compared with the wild-type cephalosporinases. We show here that ESAC expression associated with the loss of both OmpC and OmpF porins conferred in Escherichia coli a high level of resistance to ertapenem and reduced the susceptibility to imipenem. On the contrary, ESAC expressed in the OmpC- or OmpF-deficient E. coli strains or narrow-spectrum cephalosporinase expressed in the OmpC-and OmpF-deficient strain do not confer reduced susceptibility to any of the carbapenems. The production of ESAC beta-lactamase in favorable E. coli background may represent an additional mechanism of resistance to ertapenem.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin.

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.     

Role of nitric oxide in D-galactosamine-induced cell death and its protection by PGE1 in cultured hepatocytes.

Prostaglandin E(1) (PGE(1)) reduces cell death in experimental and clinical manifestations of liver dysfunction. Nitric oxide (NO) has been shown to exert a protective or noxious effect in different experimental models of liver injury. The aim of the present study was to investigate the role of NO during PGE(1) protection against D-galactosamine (D-GalN) citotoxicity in cultured hepatocytes. PGE(1) was preadministered to D-GalN-treated hepatocytes. The role of NO in our system was assessed by iNOS inhibition and a NO donor. Different parameters related to apoptosis and necrosis, NO production such as nitrite+nitrate (NO(x)) release, iNOS expression, and NF-kappaB activation in hepatocytes were evaluated. The inhibition of iNOS reduced apoptosis induced by D-GalN in hepatocytes. PGE(1) protection against D-GalN injury was associated with its capacity to reduce iNOS expression and NO production induced by D-GalN. Nevertheless, iNOS inhibition showed that protection by PGE(1) was also mediated by NO. Low concentrations of a NO donor reduced D-GalN injury with a decrease in the extracellular NO(x) concentration. High concentrations of the NO donor enhanced NO(x) concentration and increased cell death by D-GalN. The present study suggests that low NO production induced by PGE(1) preadministration reduces D-GalN-induced cell death through its capacity to reduce iNOS expression and NO production caused by the hepatotoxin.     

Photo oxidation of rice starch II. Using a water soluble photo initiator

A new photo oxidation system was established when rice starch was oxidized using UV irradiation and 4-(trimethyl ammoniummethyl) benzophenone chloride (BP2) as a photo initiator. BP2 is a water soluble photo initiator. The slurry prepared for photo oxidation contained rice starch, water and BP2 only. No oxidizing agents were added. Parameters affecting the photo oxidation process, i.e. temperature, concentration of BP2, material:liquor ratio and irradiation time were determined. The produced oxidized starch was evaluated by measuring the carboxyl content, carbonyl content and apparent viscosity. The produced photo oxidized rice starch showed sound increase in the carboxyl and carbonyl contents and sharp decrease in the apparent viscosity. The produced photo oxidized starch was tested for its suitability as a sizing agent for cotton yarns. Native starch and oxidized starch, used as a sizing agent by Misr Company of Spinning and Weaving in El-Mahalla El-Kubra (Egypt), were used for comparison. Sized cotton yarns were evaluated by measuring the tensile strength, elongation at break and percent of size removal. Cotton yarns sized using the prepared photo oxidized rice starch showed higher tensile strength, elongation at break and percent of size removal compared with native starch and oxidized starch used by Misr Company of Spinning and Weaving.