Detection and frequency of Stx2 gene in Escherichia coli O157 and O157:H7 strains isolated from sheep carcasses in Shiraz-Iran

Enterohaemorrhagic Escherichia coli constitute a subset of serotypes (E. coli O157 and some other serogroups) of Shiga toxin-producing E. coli firmly associated with severe human illnesses like bloody diarrhoea and haemolytic uraemic syndrome. Escherichia coli O157:H7 is a zoonotic pathogen. They rarely cause disease in animals, live in the intestines of healthy sheep and ruminants are recognized as their main natural reservoir, so they can contaminate meat during slaughtering practices. The purpose of this study was epidemiological survey on the occurrence of E. coli O157:H7 in healthy sheep in Shiraz-Iran. Polymerase Chain Reaction (PCR) assay was developed to detect the Stx2 gene the only bacterial factor that has been associated with more severe disease. During a period of 7 months (December 2005 to June 2006), 153 slaughtered sheep at Shiraz slaughterhouse, were randomly selected and examined for surface carriage of E. coli O157:H7 by conventional plating and Stx2 gene detection by PCR technique. E. coli O157:H7 was found in 6(3.92%) of 153 sheep. The bacteria were isolated from 5(3.34%) of 114 and 1(2.63%) of 38 sheep two or under two and more than 2 years old, respectively (p = 0.5). The contamination rate might vary depending on season, age and infection time. The higher frequency for younger animals may be due to differences in the composition of the gastrointestinal flora resulting from differences in diet. This is the first report of the presence of E. coli O157:H7 in sheep from Iran.     

Prevalence of Escherichia coli O157:H7 among diarrhoeic HIV/AIDS patients in the Eastern Cape Province-South Africa

This study investigated the prevalence of Escherichia coli O157:H7 in the stool of confirmed and non-confirmed diarrhoeic HIV/AIDS patients. Escherichia coli O157:H7 was isolated by culture-based and immunomagnetic separation from three hundred and sixty stool swabs. Identification was by conventional IMViC, 20E API and molecular techniques. Confirmed and non-confirmed diarrhoeic HIV/AIDS patients had 56.5% (74/131) and 43.5% (57/131) respectively of E. coli O157:H7. Molecular results indicated that the prevalence of E. coli O157:H7 was 12.16% (9/74) and 8.77% (5/57) from stool swabs of confirmed and non-confirmed diarrhoeic HIV/AIDS patients. Antimicrobial resistance was higher for E. coli O157:H7 isolates from stools of confirmed HIV/AIDS than it was for non-confirmed HIV/AIDS patients. Escherichia coli O157:H7 might be a silent cause of diarrhoea in HIV/AIDS patients. It is recommended that HIV/AIDS patients with diarrhoea should be screened for E. coli O157:H7 and surveillance programmes for these bacteria should be established in both urban and rural areas of South Africa.     

Involvement of triadimefon induced early ABA-dependent H2O2 accumulation in soybean against water stress

The present study showed that pretreatment of triadimefon (TDM), a triazole compound, could improve tolerance of soybean seedlings to subsequent water stress. TDM pretreatment resulted in early and late rise in superoxide dismutase (SOD) and catalase (CAT) activities, and upregulation of ascorbate (AsA) content in non-stressed and water-stressed seedlings, leading to late increase in net photosynthetic rate (P_n), late decrease in hydrogen peroxide (H₂O₂) and electrolyte leakage in stressed ones. These TDM-induced changes were blocked by application of abscisic acid (ABA) biosynthesis inhibitor tungstate, which inhibited early rise of ABA and H₂O₂ contents in non-stressed and stressed seedlings. However, ABA pretreatment overcomed the effects of this inhibitor. Application of NADPH oxidase inhibitor diphenyleneiodonium (DPI), polyamine oxidase (PAO) inhibitor 2-hydroxyethylhydrazine (2-HEH) and H₂O₂ scavenger dimethylthiourea (DMTU) prevented early TDM-induced rise of H₂O₂ content. DPI, 2-HEH and DMTU also decreased SOD, CAT and AsA levels, but did not affect ABA content during early and late phases in both seedlings pretreated with TDM. In addition, these chemicals decreased P_n, and increased H₂O₂ content and electrolyte leakage during late phase in TDM-pretreated stressed seedlings. Overall, these results indicated that TDM pretreatment alleviated adverse effects of water stress on soybean seedlings, which was at least in part, due to increase of antioxidant capacity and decrease of oxidative damage induced by early ABA-dependent H₂O₂ generation.     

Design and Synthesis of Novel Xyloketal Derivatives and Their Protective Activities against H2O2-Induced HUVEC Injury

In this work, we designed and synthesized a series of amide derivatives (1–13), benzoxazine derivatives (16–28) and amino derivatives (29–30) from xyloketal B. All 28 new derivatives and seven known compounds (14, 15, 31–35) were evaluated for their protection against H₂O₂-induced HUVEC injury. 23 and 24 exhibited more potential protective activities than other derivatives; and the EC₅₀ values of them and the leading compound 31 (xyloketal B) were 5.10, 3.59 and 15.97 μM, respectively. Meanwhile, a comparative molecular similarity indices analysis (CoMSIA) was constructed to explain the structural activity relationship of these xyloketal derivatives. This 3D QSAR model from CoMSIA suggested that the derived model exhibited good predictive ability in the external test-set validation. Derivative 24 fit well with the COMSIA map, therefore it possessed the highest activity of all compounds. Compounds 23, 24 and 31 (xyloketal B) were further to examine in the JC-1 mitochondrial membrane potential (MMP) assay of HUVECs using flow cytometry (FCM). The result indicated that 23 and 24 significantly inhibited H₂O₂-induced decrease of the cell mitochondrial membrane potential (ΔΨm) at 25 μM. Collectively, the protective effects of xyloketals on H₂O₂-induced endothelial cells may be generated from oxidation action by restraining ROS and reducing the MMP.     

Biochemical and Biological Evaluation of an L-Asparaginase from Isolated Escherichia coli MF-107 as an Anti-Tumor Enzyme on MCF7 Cell Line

Background: One of the most widely used anticancer agents is microbial L-ASNase. Herein, we assessed the biochemical and biological properties of an isolated L-ASNase from a Gram-negative bacteria strain, Escherichia coli MF-107. Methods:Using garden asparagus, we obtained several bacterial isolates. These strains were further screened for L-ASNase activity. A promising bacterial isolate was selected for L-ASNase production and subsequent purification. The molecular weight of purified L-ASNase was determined. The MTT assay was applied to assess the cytotoxic effect of the purified enzyme. Also, for caspase activity determination and the apoptotic effect of purified enzyme on in cells, we conducted a real-time PCR method. Results:The molecular weight of the enzyme was approximately 37 kDa. In the pH range of 7.5 to 8, the enzyme had considerable stability. At 35 °C, the purified L-ASNase optimum activity was recorded. The cytotoxic effect of the enzyme on treated cells was dose-dependent with an IC₅₀ value of 5.7 IU/ml. The Bax gene expression considerably raised by 5.75-fold (p < 0.001) upon L-ASNase treatment. On the other hand, the anti-apoptotic Bcl-2 gene expression showed a 2.63-fold increase compared to the control (p < 0.05). It was detected that the mRNA levels of caspase-3 and p53 were considerably upregulated (5.93 and 1.85-fold, respectively). We did not find any alternation in the caspase-8 activity of the treated cells compared to untreated cells. Conclusion:In this research, the proliferation of the breast cancer cells remarkably inhibited via the cytotoxic effect of isolated L-ASNase from microbial sources.Key Words: Apoptosis, Breast cancer, Escherichia coli, MCF7 cell line     

茶树花青素还原酶基因在大肠杆菌中的表达及优化

茶树花青素还原酶是催化非酯型儿茶素EC和EGC合成的关键酶。采用RT-PCR技术,获得了茶树花青素还原酶基因的开放阅读框,它编码含337个氨基酸的蛋白质,推测分子量为37kD,等电点为6.54;成功地将该基因重组到表达载体pET32a(+)上,并在大肠杆菌rosetta中进行原核表达;优化了原核表达中诱导时间、诱导温度、IPTG浓度、氨苄青霉素浓度,纯化出目的蛋白。HPLC检测表明,目的蛋白具有ANR酶活性。     

The Galleria mellonella-Enteropathogenic Escherichia coli Model System: Characterization of Pathogen Virulence and Insect Immune Responses

The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies using this system. We found that EPEC causes disease in G. mellonella larvae when injected intrahemocoelically but not orally. Disease manifests as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, increased pupal duration, and hemocytopenia. Disease symptoms are dose-dependent and can be used as metrics for measuring EPEC virulence in future studies. The type III secretion system was only partially responsible for EPEC virulence in G. mellonella while the majority of the virulence remains unknown in origin. EPEC elicits insect anti-bacterial immune responses including melanization, hemolymph coagulation, nodulation, and phagocytosis. The immune responses were unable to control EPEC replication in the early stage of infection (≤3 h post-injection). EPEC clearance from the hemocoel does not guarantee insect survival. Overall, this study provided insights into EPEC virulence and pathogenesis in G. mellonella and identified areas of future research using this system.     

The Lipopolysaccharide Export Pathway in Escherichia coli: Structure,Organization and Regulated Assembly of the Lpt Machinery

The bacterial outer membrane (OM) is a peculiar biological structure with a unique composition that contributes significantly to the fitness of Gram-negative bacteria in hostile environments. OM components are all synthesized in the cytosol and must, then, be transported efficiently across three compartments to the cell surface. Lipopolysaccharide (LPS) is a unique glycolipid that paves the outer leaflet of the OM. Transport of this complex molecule poses several problems to the cells due to its amphipatic nature. In this review, the multiprotein machinery devoted to LPS transport to the OM is discussed together with the challenges associated with this process and the solutions that cells have evolved to address the problem of LPS biogenesis.     

Saccharobisindole,Neoasterric Methyl Ester,and 7-Chloro-4(1H)-quinolone: Three New Compounds Isolated from the Marine Bacterium Saccharomonospora sp.

Analysis of the chemical components from the culture broth of the marine bacterium Saccharomonospora sp. CNQ-490 has yielded three novel compounds: saccharobisindole (1), neoasterric methyl ester (2), and 7-chloro-4(1H)-quinolone (3), in addition to acremonidine E (4), pinselin (5), penicitrinon A (6), and penicitrinon E (7). The chemical structures of the three novel compounds were elucidated by the interpretation of 1D, 2D nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) data. Compound 2 generated weak inhibition activity against Bacillus subtilis KCTC2441 and Staphylococcus aureus KCTC1927 at concentrations of 32 μg/mL and 64 μg/mL, respectively, whereas compounds 1 and 3 did not have any observable effects. In addition, compound 2 displayed weak anti-quorum sensing (QS) effects against S. aureus KCTC1927 and Micrococcus luteus SCO560.     

Novel Antioxidant Collagen Peptides of Siberian Sturgeon (Acipenser baerii) Cartilages: The Preparation,Characterization, and Cytoprotection of H2O2-Damaged Human Umbilical Vein Endothelial Cells (HUVECs)

For making full use of aquatic by-products to produce high value-added products, Siberian sturgeon (Acipenser baerii) cartilages were degreased, mineralized, and separately hydrolyzed by five kinds of proteases. The collagen hydrolysate (SCH) generated by Alcalase showed the strongest 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) and hydroxide radical (HO·) scavenging activity. Subsequently, thirteen antioxidant peptides (SCP1-SCP3) were isolated from SCH, and they were identified as GPTGED, GEPGEQ, GPEGPAG, VPPQD, GLEDHA, GDRGAEG, PRGFRGPV, GEYGFE, GFIGFNG, PSVSLT, IELFPGLP, LRGEAGL, and RGEPGL with molecular weights of 574.55, 615.60, 583.60, 554.60, 640.64, 660.64, 885.04, 700.70, 710.79, 602.67, 942.12, 714.82, and 627.70 Da, respectively. GEYGFE, PSVSLT, and IELFPGLP showed the highest scavenging activity on DPPH· (EC₅₀: 1.27, 1.05, and 1.38 mg/mL, respectively) and HO· (EC₅₀: 1.16, 0.97, and 1.63 mg/mL, respectively), inhibiting capability of lipid peroxidation, and protective functions on H₂O₂-damaged plasmid DNA. More importantly, GEYGFE, PSVSLT, and IELFPGLP displayed significant cytoprotection on HUVECs against H₂O₂ injury by regulating the endogenous antioxidant enzymes of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to decrease the contents of reactive oxygen species (ROS) and malondialdehyde (MDA). Therefore, the research provided better technical assistance for a higher-value utilization of Siberian sturgeon cartilages and the thirteen isolated peptides—especially GEYGFE, PSVSLT, and IELFPGLP—which may serve as antioxidant additives for generating health-prone products to treat chronic diseases caused by oxidative stress.     

A review of the biology and control of selected weed species in the genus Oxalis: O. stricta L., O. latifolia H.B.K. and O. pes-caprae L.

This review considers the distribution, weed problems, biology and control of Oxalis stricta, O. latifolia and O. pes-caprae. The rhizomatous O. stricta is a persistent seed-producing weed which invades horticultural crops in North America. Chemical control is generally most effective in situations where soil-applied residual herbicides can be used; in turfgrass however, foliage-applied herbicides provide suppression only. O. latifolia is widely distributed in a range of annual and perennial crops growing in temperate and tropical locations. This pernicious perennial weed reproduces exclusively by asexual structures, underground bulbs, stolons and tubers. The complexities of the perennating system have rendered ineffective a wide range of herbicide families as a means of restricting the spread of O. latifolia. Similarly, cultural methods of control have not been successful. O. pes-caprae also grows and perennates from bulbs. This weed is essentially a problem in cereal crops in Southern Australia and Mediterranean fruit orchards. Cultural and chemical control difficulties and inadequacies are similar to those in O. latifolia. Future research strategies involving weed physiology, herbicide action and cultural control including biological control are discussed in relation to obtaining improved control methods for the selected Oxalis species.