Mohamed F. Ghaly

Membrane-bound Quinoprotein D-Arabitol Dehydrogenase of Gluconobacter suboxydans IFO 3257: A Versatile Enzyme for the Oxidative Fermentation of Various Ketoses

Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purifiy ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82kDa (subunit I) and 14kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.

Characterization of Streptomyces isolates causing colour changes of mural paintings in ancient Egyptian tombs

Paintings in ancient Egyptian tombs often suffer colour changes due to microbial growth and colonization. Streptomyces strains were isolated from mural paintings of Tell Basta and Tanis tombs (East of Nile Delta, Egypt) and were identified using biochemical and molecular methods. The16S rDNA sequences data indicated that isolated strains were closely related to S. coelicolor, S. albidofuscus, S. ambofaciens, S. canarius, S. parvullus, S. corchorusii, S. albidofuscus and S. nigrifaciens. It could be shown that Streptomyces strains are involved on a large scale in the colour changes of paintings and stone support by producing a wide range of metabolites such as acids (oxalic, citric and sulphuric acids), biopigments of melanin, carotenoids, and hydrogen sulphide.

Production and prebiotic activity of exopolysaccharides derived from some probiotics

Objective The aim of this study was to focus on exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii bulgaricus, Lactobacillus helveticus or Lactobacillus casei and their use as a prebiotic for Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus acidophilus or Bifidobacterium bifidum. Materials and methods Optimization of culture conditions using different carbon and nitrogen sources and different temperature, pH and incubation periods for maximum EPS production was studied. Results and conclusion It was found that the best conditions were as follows: the use of sucrose (20%) instead of glucose in MRS medium, incubation at pH 7.0 and temperature 37°C for 72 h incubation under anaerobic conditions to give the highest EPS yield; a yield of 13.99 g/l was recorded in case of L. helveticus when grown on the aforementioned optimized conditions. It was found that L. delbrueckii bulgaricus EPS has the highest prebiotic indices (I), varying from 7.9 to 10.1. In contrast, L. helveticus and L. casei EPSs have the lowest prebiotic indices (I), varying from 1.4 to 2.4.

Diagnostic performances of hepatitis C virus-NS4 antigen in patients with different liver pathologies.

BACKGROUND AND AIMS Hepatitis C virus (HCV) has emerged as the major pathogen of liver disease worldwide. The aim of this study was to quantitate and evaluate the performance of HCV-NS4 antigen as an alternative approach for confirmation of viremia. METHODS Detection of HCV-NS4 was assessed in 883 patients with chronic hepatitis C. Areas under the ROC curves (AUC) were used to assess and compare diagnostic accuracy of ELISA for HCV-NS4 with quantitative HCV-RNA as a gold standard. RESULTS HCV-NS4 was identified at 27 kDa using Western blot. AUC for HCV-NS4 detection was 0.95 for all patients with different liver pathologies: 0.93 for liver fibrosis (LF), 0.95 for liver cirrhosis (LC) and 0.98 for hepatocellular carcinoma (HCC). The mean ± SD (μg/mL) of HCV-NS4 in LF was 94.2 ± 55.6; in LC was 99.3 ± 64.8 and in HCC was 124.9 ± 70.3. CONCLUSIONS HCV-NS4 antigen detection using ELISA is a reliable test in the confirmation of HCV infection.

Perinatal transmission of hepatitis c antigens: envelope 1, envelope 2 and non-structural 4

Abstract Background: Perinatal exposure to hepatitis C virus (HCV) antigens during pregnancy may affect the developing immune system in the fetus. We aimed to study the perinatal transmission of HCV structural and non-structural antigens. Methods: Sera from 402 pregnant mothers were tested for anti-HCV antibody and HCV RNA. HCV antigens were determined in sera from 101 HCV-infected mothers and their cord blood. Results: In both serum and cord blood samples, HCV NS4 (non-structural 4) at 27 kDa, E1 (envelope 1) at 38 kDa and E2 (envelope 2) at 40 kDa were identified, purified and quantified using western blotting, electroelution and ELISA. Maternal sera and neonate cord blood samples had similar detection rates for NS4 (94.1%), E1 (90.1%) and E2 (90.1%). The mean maternal serum levels (optical density, OD) of HCV NS4 (0.87 ± 0.01), E1 (0.86 ± 0.01) and E2 (0.85 ± 0.01) did not differ significantly (p > 0.05) from those of neonatal cord blood (0.83 ± 0.01, 0.87 ± 0.01 and 0.85 ± 0.01, respectively). Also, strong correlations (p < 0.0001) were shown between sera and cord blood sample levels of HCV NS4, r = 0.77; E1, r = 0.76 and E2, r = 0.80. The vertical transmission of these antigens in vaginal delivery did not differ significantly (p > 0.05) from those in caesarean section. Conclusions: These findings indicate that vertical transmission of HCV NS4, E1 and E2 antigens was very high. Thus, exposure to these antigens may influence the developing immune responses to natural infection or future vaccination.

Circulating levels of collagen III and MMP-1 in patients with chronic hepatitis C co-infected with hepatitis B virus

Abstract Background: There is controversial data in the literature about the characteristics and features of dual hepatitis B and hepatitis C infection. This work is concerned with estimating the extent to which HBV could influence circulating levels of hepatitis C viral nonstructural-4 (HCV-NS4) in addition to some direct fibrosis markers in chronic hepatitis C. Methods: Thirty-eight HCV mono-infected and 87 HCV/HBV co-infected patients constituted this study. Western-blot and ELISA were used for identifying HCV-NS4, hepatitis B surface antigen (HBsAg), collagen III and matrixmetalloproteinase-1 (MMP-1) in patients’ sera. Results: Hepatitis B surface antigen (HBsAg) provided area under curve (AUC) of 0.97 for identifying HBV-patients with 89% sensitivity and 94% specificity, while HCV-NS4 antigen provided an AUC of 0.95 for identifying HCV-patients with 89% sensitivity and absolute specificity (100%). In general, patients with significant fibrosis (F2–F4) showed significantly higher concentration of collagen III (P = 0.009) and lower concentrations of MMP-1 (P = 0.007) when compared to patients with minimal fibrosis (F1). However, HCV/HBV co-infected patients with F1 and F2–F4 did not show any significant difference (P > 0.05) from HCV mono-infected patients with respect to HCV-NS4, collagen III and MMP-1. These results indicate that HBV does not influence the rate of HCV-NS4 synthesis and the deposition of extracellular matrix in HCV/HBV co-infected patients and subsequently does not affect the progression rates of hepatic fibrosis. Conclusion: HCV/HBV co-infected and HCV- mono-infected patients had similar clinical characteristics and there is no effect of HBV co-infection on the progression rates of liver fibrosis in chronic hepatitis C patients.