Towards β-globin gene-targeting with integrase-defective lentiviral vectors

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.     

Biodegradation pathway and detoxification of the diazo dye Reactive Black 5 by Phanerochaete chrysosporium

The in vivo biodegradation of the diazo dye Reactive Black 5 (RB5) by Phanerochaete chrysosporium immobilised on cubes of nylon sponge and on sunflower-seed shells (SS) in laboratory-scale bioreactors was investigated. The SS cultivation led to the best results with a decolouration percentage of 90.3% in 72 h for an initial RB5 concentration of 100 mg/L. It was found that the addition of 0.4 mM veratryl alcohol (VA) into the medium considerably increased the decolouration rate in SS cultivation. However, the addition of VA had no effect in the nylon cultivation. Thin layer chromatography (TLC) revealed that RB5 was transformed into one metabolite after 24 h. UV-vis spectroscopy and Fourier Transform Infrared (FT-IR) also confirmed the biodegradation of RB5. Toxicity of RB5 solutions before and after fungal treatment was assayed using Sinorhizobium meliloti as a sensitive soil microorganism. P. chrysosporium transformed the toxic dye RB5 into a non-toxic product.     

Prevalence of Cryptosporidium-like infection in one-humped camels (Camelus dromedarius) of northwestern Iran

Cryptosporidium is a ubiquitous enteropathogen protozoan infection affecting livestock worldwide. The present study was carried out to determine the prevalence of Cryptosporidium infection in different age groups of dromedary camels in northwestern Iran from November 2009 to July 2010. A total number of 170 fecal samples were collected and examined using modified Ziehl-Neelsen (MZN) staining under light microscope. Examination of stained fecal smears revealed that 17 camels (10%) were positive for Cryptosporidium-like. The prevalence of Cryptosporidium-like was significantly higher in camel calves (< 1 years old) (20%) than other age groups, in which the diarrhoeic calves had the prevalence of 16%. In adult camels the prevalence was 6.5%. There was no significant difference in the prevalence of Cryptosporidium-like between male and female camels. It is concluded that Cryptosporidium infection is a problem in camel husbandry and could be of public health concern in the region.     

Performance evaluation of potent phosphate solubilizing bacteria in potato rhizosphere

Three phosphate solubilizing bacterial isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 were assessed for mutual relationships among them, competitiveness with soil microorganisms and associations with plant root using luxAB reporter genes for follow-up studies. Synergism between either P. agglomerans or M. laevaniformans, as acid-producing bacteria, and P. putida, as a strong phosphatase producer, was consistently observed both in liquid culture medium and in root rhizosphere. All laboratory, greenhouse and field experiments proved that these three isolates compete well with naturally occurring soil microorganisms. Consistently, the combinations of either P. agglomerans or M. laevaniformans strains with Pseudomonas putida led to higher biomass and potato tuber in greenhouse and in field trials. It is conceivable that combinations of an acid- and a phosphatase-producing bacterium would allow simultaneous utilization of both inorganic and organic phosphorus compounds preserving the soil structure.     

In silico prediction of exposure amino acid sequences of outer inflammatory protein A of Helicobacter pylori for surface display on Eschierchia coli

BACKGROUND:

Outer inflammatory protein A (OipA) is an outer membrane protein of Helicobacter pylori that is involved in inducing IL-8 and intracellular signaling. In this study, we have predicted exposure amino acid sequences of OipA for insertion in permissive sites of CstH subunit of Eschierchia coli CS3 pilli for bacterial surface display.

MATERIALS AND METHODS:

Databases: National Center for Biotechnology Institute and Protein Data Bank. Servers: PHD, SABLE, GOR 4, SignalP3.0, TBBpred, PRODIV-TMHMM, TMRPres2D, CPH Models, PHYRE, GETAREA, VADAR, Pep state and pep window. Software: Swiss PDB viewer and Discovery studio.

RESULTS:

In silico prediction of exposure amino acid sequences of OipA led to detection of six sequences of amino acid, 76-87, 106-112, 170-182, 222-230, 242-258, and 278-290. These sequences inserted between amino acid sequences 66-67, 100-101, and 109-110 of CstH that were predicted by Eskandari et al. as permissive sites of CstH.

CONCLUSION:

OipA has the ability to induce IL-8 from gastric epithelial cells and some papers are mentioned that this outer membrane protein involve to attachment and intracellular signaling. Receptor of OipA and adhesion motifs on this protein is unknown. Detection of exposure motifs aids to recognition of adhesion motifs and receptor of OipA on gastric epithelial cells. In this study, we have predicted exposure amino acid sequences for insert to subunit CstH of CS3 pilli E. coli for surface display.     

Cloning and Characterization of a Plasmid Encoded ACC Deaminase from an Indigenous Pseudomonas fluorescens FY32

In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into α-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5α proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process.     

Immobilization of recombinant nanobiofiber CS3 fimbriae onto alginate beads for improvement of cadmium biosorption

A cell surface display system with metalbinding properties was previously developed using CS3 fimbriae, which are hollow tubes 20 nm-thick and 2 nm in diameter. In this study, hybrid CS3 pili were separated from recombinant Escherichia coli and entrapped in calcium alginate gel beads in order to improve their stabilization and also adsorption of heavy metals. The surface morphology of the gel beads containing pili was investigated by scanning electron microscopy (SEM). Immunofluorescence microscopy was employed to confirm the attachment of nanobiofibers to the alginate beads. The effects of three variables (sodium alginate concentration, protein to alginate mass ratio, and bead size) at two levels each on Cd²⁺ biosorption efficiency were investigated by full factorial experimental design. A second-order polynomial equation modeled the design space for the process response of cadmium removal capacity. The optimal values of the factors were obtained as follows: 1% sodium alginate concentration, 0.25 protein to alginate mass ratio, and a 6 mm bead size. Under these conditions, Cd²⁺ was adsorbed at 45.45 mg/g to the nanobiofiber. The results indicate that the immobilized recombinant hybrid CS3 pili may be an appropriate biosorbent for removal of heavy metals from polluted aquatic environments.     

Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacterxylinus in E. coli Nissle 1917

Bioprocess and Biosystems Engineering - Based on cellulose biosynthesis pathway of Gluconacetobacterxylinus BPR2001 and E. coli Nissle 1917, bcsA and bcsB genes have been selected and...     

Heat-Induced Production of Human Growth Hormone by High Cell Density Cultivation of Recombinant Escherichia Coli

The temperature-induced, over-expression of the human growth hormone gene in a recombinant E. coli during high cell density cultivation is reported. Human growth hormone (hGH) production and stability were tested under different heat shock conditions. Cell densities were 25 and 60 g l(-1) in a pH-stat fed-batch mode in defined and complex medium, respectively, and the fermentation time was decreased from 41 to 32 h. hGH was produced at 2 g l(-1) in complex medium. By using glycerol as main carbon source in the complex medium with exponential feeding, cell density and hGH production were increased to 100 g l(-1) and 2.7 g l(-1), respectively.