Isolation and characterization of biocides resistant bacteria from dental unit water line biofilms

Six biocides resistant isolates were isolated from dental unit water lines (DUWL) in Pakistan. All isolates could tolerate 150 μg ml^–1 of biocides (5.25% sodium hypocholrite, 35% H₂O₂, 4% tween 20, 1% povidine iodine, 0.2% chlorohexidine gluconate, 1% ethylene di‐amino tetra acetic acid and 1% phenol) on l‐agar and 100 μg ml^–1 in l‐broth. The growth rate of all isolates was determined by generating growth curves at 37 °C for 48 h. The isolates were found to differ in growth rates with lag phase varying from (4–6 h) in biocides supplemented media compared to 2–4 h in biocides free medium. They have wide temperatures (24–42 °C) and pH (5–9) ranges. Traditional ways of identification of bacteria by phenotypic characteristics were accomplished by phenotypic and biochemical characterization. Heavy metals and antimicrobial susceptibility tests indicated that all isolates examined were resistant to trimethoprim, chloramphenicol while sensitive to HgCl₂ and Pb (NO₃)₂. Almost all isolates were opportunistic pathogens. The 16S rRNA‐encoding genes from these six isolates were sequenced to confirm the identity of these isolates. 5 different genera (Bacillus, Achromobacter, Pseudomonas and Klebsiella) of bacteria were identified by 16S rDNA genes amplified from genomic DNA of biocides resistant DUWL biofilm isolates. Analysis of 16S rDNA genes revealed a much more clear identification of microrganisms than culture methods. However, different species of the same genera can have the same 16S rRNA gene sequence but are different due to phenotypic differences or different clinical manifestations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport

1 Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide galanin in human erythrocytes in vitro. 2 The potencies of nootropic drugs in opposing scopolamine-induced memory loss correlate with their potencies in antagonising pentobarbital inhibition of erythrocyte glucose transport in vitro (P<0.01). Less potent nootropics, D-levetiracetam and D-pyroglutamate, have higher antagonist Ki's against pentobarbital inhibition of glucose transport than more potent L-stereoisomers (P<0.001). 3 Piracetam and TRH have no direct effects on net glucose transport, but competitively antagonise hypnotic drug inhibition of glucose transport. Other nootropics, like aniracetam and levetiracetam, while antagonising pentobarbital action, also inhibit glucose transport. Analeptics like bemigride and methamphetamine are more potent inhibitors of glucose transport than antagonists of hypnotic action on glucose transport. 4 There are similarities between amino-acid sequences in human glucose transport protein isoform 1 (GLUT1) and the benzodiazepine-binding domains of GABAA (gamma amino butyric acid) receptor subunits. Mapped on a 3D template of GLUT1, these homologies suggest that the site of diazepam and piracetam interaction is a pocket outside the central hydrophilic pore region. 5 Nootropic pyrrolidone antagonism of hypnotic drug inhibition of glucose transport in vitro may be an analogue of TRH antagonism of galanin-induced narcosis.     

Mercury-Induced Chromosomal Damage in Wild Fish (Dicentrarchus labrax L.) Reflecting Aquatic Contamination in Contrasting Seasons

Ria de Aveiro (mainly Laranjo basin, Portugal) has been subjected to mercury contamination from a chlor-alkali plant, currently presenting a well-described mercury gradient. This study aimed to assess mercury genotoxicity in this area by measuring the frequency of erythrocytic nuclear abnormalities (ENA) in the European sea bass (Dicentrarchus labrax), addressing the relation with total mercury concentration in the blood and the modulatory role of seasonal variables. Fish were collected, in warm and cold periods, at three locations differing in their distances to the main mercury source: reference (R), moderately (M), and highly (H) contaminated sites. Genotoxicity was detected in both degrees of contamination (M and H) and in both periods of the year (warm and cold), which is in line with the greater levels of mercury measured in fish blood. No significant seasonal variations were observed for mercury bioaccumulation or ENA frequency. The apparent low imperviousness of ENA frequency to seasonal factors reinforced its consistency as a genotoxicity biomarker, thus enabling a clearer identification of cause-and-effect relationships. Overall, the results reflected a serious environmental risk to native ichthyofauna at Laranjo basin due to mercury contamination, showing a potential of mercury to induce genetic damage in fish blood cells through clastogenic and/or aneugenic actions.     

Effective utilization of magnetic nano-coupled cloned β-xylanase in saccharification process

The β-xylanase gene (DCE06_04615) with 1041 bp cloned from Thermotoga naphthophila was expressed into E. coli BL21 DE3. The cloned β-xylanase was covalently bound to iron oxide magnetic nanoparticles coated with silica utilizing carbodiimide. The size of the immobilized MNPs (50 nm) and their binding with β-xylanase were characterized by Fourier-transform electron microscopy (FTIR) (a change in shift particularly from C–O to C–N) and transmission electron microscopy (TEM) (spherical in shape and 50 nm in diameter). The results showed that enzyme activity (4.5 ± 0.23 U per mL), thermo-stability (90 °C after 4 hours, residual activity of enzyme calculated as 29.89% ± 0.72), pH stability (91% ± 1.91 at pH 7), metal ion stability (57% ± 1.08 increase with Ca²⁺), reusability (13 times) and storage stability (96 days storage at 4 °C) of the immobilized β-xylanase was effective and superior. The immobilized β-xylanase exhibited maximal enzyme activity at pH 7 and 90 °C. Repeated enzyme assay and saccharification of pretreated rice straw showed that the MNP-enzyme complex exhibited 56% ± 0.76 and 11% ± 0.56 residual activity after 8 times and 13 times repeated usage. The MNP-enzyme complex showed 17.32% and 15.52% saccharification percentage after 1^st and 8^th time usage respectively. Immobilized β-xylanase exhibited 96% residual activity on 96 days'' storage at 4 °C that showed excellent stability.

The β-xylanase gene (DCE06_04615) with 1041 bp cloned from Thermotoga naphthophila was expressed into E. coli BL21 DE3.     

Interferon α2–Thymosin α1 Fusion Protein (IFNα2–Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2–thymosin α1 fusion protein (IFNα2–Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2–Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2–Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2–Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2–Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.