ORIGINAL_ARTICLE
The effects of novel mutations in A1 domain of human coagulation factor VIII on its secretion level in cultured mammalian cells
Inefficient secretion of the human coagulation factor (hFVIII) in mammalian expression systems is one ofthe main causes of the hFVIII low expression level, attributed to its interaction with a chaperone known asBiP/GRP78. In order to improve secretion efficiency of the hFVIII, based on the higher secretion level of theporcine FVIII and analysis of the hFVIII A110 region, that inhibits its secretion, function of three novel Bdomain deleted hFVIII mutant including; two singlemutants (Leu299Phe and Phe309Thr) and a doublemutant (Tyr323His/Lys325Arg) were examined in three mammalian cell lines (HEK-293T, COS, CHO) for the hFVIII secretion efficiency. The double-mutant construct displayed the highest hFVIII expression level,about seven-fold as much the base-line. The doublemutant hFVIII was biologically active and its inactivationpatterns by EDTA and heat was similar to that of the non-mutant hFVIII. Semi-quantitative RT-PCRresults showed the highest mRNA level for the doublemutant hFVIII. Both of the mutated residues in the double-mutant are located in a hydrophobic heptamer (320MEAYVKV326) that seems to be involved in Bipbinding activity. None of the L299F and F309T hFVIII mutants exhibited improved secretion. This result hasprovided convincing evidence for the increasing effect of the double-mutant on the hFVIII secretion and transcription efficiencies.
https://www.ijbiotech.com/article_7115_57ca85e9ba487bfd6809a796c2f40797.pdf
2010-07-01
139
149
Hemophilia A
human coagulation Factor VIII
BiP
A110 region
Secretion
mammalian expression system
Gholam Ali
Kardar
kardar51@yahoo.com
1
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
AUTHOR
Alireza
Zomorodipour
zomorodi1@gmail.com
2
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
LEAD_AUTHOR
Mostafa
Moin
3
Immunology, Asthma and Allergy Research Institute, Children’s Medical Center Hospital, Tehran University of Medical Sciences, P.O. Box 14185-863, Tehran, I.R. Iran.
AUTHOR
Zahra
Pourpak
4
Immunology, Asthma and Allergy Research Institute, Children’s Medical Center Hospital, Tehran University of Medical Sciences, P.O. Box 14185-863, Tehran, I.R. Iran.
AUTHOR
Mehdi
Sadeghi
m-sadeghi@ibb.ut.ac.ir
5
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
AUTHOR
Fariba
Ataei
6
National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
Loss of chloroplast trnLUAA intron in two species of Hedysarum (Fabaceae): evolutionary implications
Previous studies have indicated that in all land plants examined to date, the chloroplast gene trnLUAA isinterrupted by a single group I intron ranging from 250 to over 1400 bp. The parasitic Epifagus virginiana haslost, however, the entire gene. We report that the intron is missing from the chloroplast genome of twoarctic species of the legume genus Hedysarum (H. alpinum, H. boreale). DNA sequencing of the trnL geneand trnL-trnF intergenic spacer (trnL-F), as well as portion of trnF exon in these species confirms theabsence of trnL intron and shows that it has been deleted from the gene precisely along establishedexon/intron splicing sites. Phylogenetic analysis of trnL-F sequence data revealed that they are closelyrelated species. This indicates that the intron was lost from the chloroplast genome before the divergence ofthe two Hedysarum species. It is concluded that this rare genomic structural mutation may have occurredonce during the evolution of land plants.
https://www.ijbiotech.com/article_7116_54b2944f2ee2b2ffb627d81738417f46.pdf
2010-07-01
150
155
chloroplast DNA
Fabaceae
Hedysarum
Structural mutation
trnL UAA intron loss
Atefeh
Amirahmadi
1
Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, I.R. Iran.
AUTHOR
Shahrokh
Kazempour Osaloo
2
Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, I.R. Iran.
LEAD_AUTHOR
Ali Asghar
Maassoumi
maassoumi@yahoo.com
3
2Department of Botany, Research Institute of Forests and Rangelands, P.O. Box 13185-116, Tehran, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
Microsatellite isolation and characterization in pomegranate (Punica granatum L.)
Development of microsatellite markers has been an increasing trend in crop genetic studies because oftheir applicability in breeding programs. Here we report the development of inter simple sequencerepeat (SSRs) in pomegranate (Punica granatum L.) using an enrichment method that makes use of magneticbeads. Enriched genomic libraries with AG and ATG microsatellite motifs were constructed, and 60 positive clones were detected by a colony PCR technique, of which 43 clones showed high-quality sequences. Out of these, 32 (74.4%) contained microsatellite sequences and 25 primer pairs were designed, of which 11 (44%) revealed polymorphisms, 12 (48%) showed monomorphic patterns and 2 (8%) generated poor amplification on a set of 20 pomegranate genotypes. Eleven microsatellite primers (two of them amplified two loci) were selected to assess polymorphism in the set of genotypes. There were 44 alleles amplified over 13 loci, with an average of 3.38 alleles per locus. The mean polymorphism information content (PIC) value was 0.433 over 13 loci, which shows that the majority of the microsatellite loci are highly informative. Cluster analysis was able to separate genotypes based on their geographical distribution and type (i.e., wild or domestic). This study shows the isolation efficiency of the magnetic beads technique, the abundance of microsatellites in pomegranate, and their potential application in pomegranate genome mapping and genotyping.
https://www.ijbiotech.com/article_7117_f234c80076e3ac270520f76fe49aabdc.pdf
2010-07-01
156
163
Pomegranate
Microsatellite
genetic diversity
SSR-enrichment libraries
Saeideh
Ebrahimi
saeideh.ebrahimi@gmail.com
1
Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, I.R. Iran.
AUTHOR
Badraldin Ebrahim
Sayed-Tabatabaei
2
Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, I.R. Iran.
LEAD_AUTHOR
Bahram
Sharifnabi
sharifna@cc.iut.ac.ir
3
Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
A new changeable bioreactor for detection of organophosphate in a flow-through system
A flow-through biosensor consisting of a fixed bed bioreactor was employed to detect the insecticideparaoxon. Based on the inhibition of organophosphorous insecticide to the enzymatic activity of acetylcholinesterase (AChE), using paraoxon as a model compound, the condition for detection of the insecticide were optimized. The influence of enzyme loading on the packing surface was studied and AChE loading was set at 0.36 U/cm2 for subsequent studies.Maximum value of absorbance response occurred at the residence time in bioreactor of 5 min, that was chosen as the optimal residence time. This flow-through system gave a linear response (R2 = 0.9869) toacetylthiocholine iodide (ATChI) at concentrations of 0.050 to 1 mM. Under appropriate conditions, the inhibition of paraoxon was proportional to its concentration in two ranges, from 0.25 to 25 mg/l and 25 to 60 mg/l and the detection limit for paraoxon was 7.3×10-8 M.The incubation time was 14 min. These results demonstrate that silicate-multiwall carbon nanotube(MWCNT) sol film is very efficient for retaining the activity of AChE with a good long-term stability.
https://www.ijbiotech.com/article_7118_45effd30203761ed3446e2183b5c699c.pdf
2010-07-01
164
171
Flow-through system
Biosensor
Acetylcholinesterase
Bioreactor
Silicate sol-gel
Multiwall Carbon Nanotube
Bahman
Ebrahimi
1
Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran, I.R. Iran.
AUTHOR
Seyed Abbas
Shojaosadati
shoja@modares.ac.ir
2
Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran, I.R. Iran.
AUTHOR
Seyyed Mohammad
Mousavi
3
Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
Polymorphism in prolactin and PEPCK-C genes and its association with economic traits in native fowl of Yazd province
The objective of the present study was to investigate the polymorphism of prolactin promoter and cytosolicphosphoenol pyruvate carboxykinase (PEPCK-C) intron 3 to exon 3 regions, and its association with economictraits in native fowl of Yazd province. These traits consisted of body weight at 8 (BW8) and 12 (BW12) weeks of age, age at sexual maturity (ASM), weight at sexual maturity (WSM), mean egg weight at28, 30, and 32 weeks (MEW), and the number of eggs during the first 12 weeks of laying period (EN). Bloodsamples were collected from 159 pedigreed fowl at native fowl breeding center of Yazd province, and DNAwas extracted from the samples according to saltingout protocol. PCR amplification together with restrictionfragment length polymorphism was used to identify different genotypes of prolactin and PEPCK-Cgenes. The effect of prolactin genotypes on economic traits was analyzed using general linear model. A 24-bp indel (insertion or deletion) at nucleotide position (np) 358 was identified, but no polymorphism wasfound for PEPCK-C. Based on our results, the frequency of I and D alleles were 0.761 and 0.239,respectively. Frequencies of II, ID and DD genotypes were 0.566, 0.389 and 0.044, respectively. GenotypesII and ID were significantly associated with incresased EN (P<0.01). Meanwhile, the genotypes of the 24-bpindel site were not significantly associated with BW8, BW12, ASM, WSM and MEW (P> 0.05). The results ofcurrent study showed that using information of genes related to egg production could be used to improve theperformance of native fowl of Yazd province.
https://www.ijbiotech.com/article_7114_37bc94f282899ee6b88e429857bec692.pdf
2010-07-01
172
177
Native fowl
pepck-c gene
prolactin gene
Polymorphism
egg production
Hakimeh
Emamgholi Begli
1
Department of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box 386, Gorgan, I.R. Iran.
AUTHOR
Saeed
Zerehdaran
zereh2@gau.ac.ir
2
Department of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box 386, Gorgan, I.R. Iran.
LEAD_AUTHOR
Saeed
Hassani
hasani@gau.ac.ir
3
Department of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box 386, Gorgan, I.R. Iran.
AUTHOR
Mokhtar
Ali Abbasi
4
Animal Science Research Institute of Iran, P.O. Box 3146618361, Karaj, I.R. Iran 3Gonbad Higher Education Center, P.O. Box 163, Gonbad, I.R. Iran.
AUTHOR
Alireza
Khan Ahmadi
5
Animal Science Research Institute of Iran, P.O. Box 3146618361, Karaj, I.R. Iran 3Gonbad Higher Education Center, P.O. Box 163, Gonbad, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
Characteristics of different brewer’s yeast strains used for non-alcoholic beverage fermentation in media containing different fermentable sugars
Fermentation characteristics of four strains of brewer's yeast (Saccharomyces cerevisiae strain 70424, S.rouxii strain 2535, S. rouxii strain 2531 and Saccharomyces ludwigii strain 3447) in Yeast Moldbrothcontaining four different fermentable sugars (glucose, fructose, maltose, or sucrose) were studied. Theaim was to consider the suitability of different strain/sugar treatments for the production of non-alcoholicbeer as well as to devise treatments resulting in greatest growth rate of yeast cells. Experimentalparameters were yeast cell growth, ethanol production, pH drop, and changes in fermenting media attenuation(°Pl), during a 48 h fermentation period. Fermentation was performed at 24°C using periodic aeration practice. For S. cerevisiae, the greatest growth rate was achieved in presence of sucrose. The maximum and minimum ethanol contents at the end of fermentation were related to sucrose- (0.94% V/V) and glucose-containing (0.4% V/V) treatments, respectively. In the case of S. ludwigii, fructose stimulated the highest growth rate and the maximum and minimum ethanol contents at the end of fermentation were observed in sucrose- (0.49%), and maltose-containing (0.04%) treatments, respectively. For S. rouxii 2535, highest growth rate was observed in the presence of fructose/glucose. The maximum and minimum ethanol contents belonged to the fructose/glucose- (~ 0.40) and maltose/sucrose-containg (~ 0.01%) treatments, respectively. In the case of S. rouxii 2531, glucose and to lesser extent, fructose led to the highest growth rate and the maximum and minimum ethanol contents were observed in glucose (0.01%) and maltose/sucrose (0.00%) treatments, respectively. Applying different strains of Saccharomyces in presence of different types of sugars caused various fermentation characteristics especially with regard to growth rate and ethanol production.
https://www.ijbiotech.com/article_7119_dc2e06887577e221a9fecbf860c615e2.pdf
2010-07-01
178
185
Beer
Brewer's yeast
Ethanol
Saccharomyces
sugar
Sarah
Sohrabvandi
1
Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran, Postal code 31587-77871, Karaj, Iran.
AUTHOR
Seyyed Hadi
Razavi
2
Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran, Postal code 31587-77871, Karaj, Iran.
LEAD_AUTHOR
Seyyed Mohammad
Mousavi
3
Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran, Postal code 31587-77871, Karaj, Iran.
AUTHOR
Amir Mohammad
Mortazavian
4
Department of Food Science and Technology, Faculty of Nutrition Sciences, Food Science Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, P.O. Box 19395-4741, Tehran, Iran.
AUTHOR
ORIGINAL_ARTICLE
Cloning of EprA1 gene of Aeromonas hydrophila in Lactococcus lactis
Bacterial-based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as vaccination strategies. Developments in genetic engineering have given Gram-positive lacticacid bacteria (LAB) the advantage of being used as a host expression system for antigen delivery to inducethe immune response. A fragment containing the full length of the “eprA1” gene, encoding a temperaturestable metalloprotease of Aeromonas hydrophila isolated from fish was amplified by polymerase chain reaction (PCR) using the genomic DNA of this bacterium as template. The amplified 1038 bp fragment was digested by PstI and HindIII, followed by ligation into the corresponding site on the pNZ8048 plasmid. The ligated DNA was then transformed into Lactococcus lactis NZ9000 cells by electroporation method.Verification of cloning was carried out using restriction enzyme digestion and DNA sequencing. Gel electrpophoresis technique also detected the expression of the recombinant protease protein. The successfulcloning and expression of the eprA1 gene into L. lactis can be developed as a useful and safe system to control A. hydrophila infections in fish.
https://www.ijbiotech.com/article_7120_464477b340289dbffcc78f80c3a5c492.pdf
2010-07-01
193
198
Aeromonas hydrophila
Protease
Lactococcus lactis
Live vaccine
fish
Hosseinali
Sasan
1
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, P.O. Box: 76169141111, Kerman, I.R. Iran.
LEAD_AUTHOR