Role of GABAB Receptor and L-Arg in GABA-Induced Vasorelaxation in Non-diabetic and Streptozotocin-Induced Diabetic Rat Vessels

Background:

Hypertension is considered an independent risk factor for cardiovascular mortality in diabetic patients. The present study was designed to determine the role of gamma amino butyric acid B (GABA_B) receptor and L-arginine (L-Arg) in GABA-induced vasorelaxation in normal and streptozotocin-induced diabetic rat vessels.

Methods:

Diabetes was induced by a single i.p. injection of streptozotocin (STZ, 60 mg/kg). Eight weeks later, superior mesenteric arteries of all groups were isolated and perfused according to the McGregor method.

Results:

Baseline perfusion pressure of STZ diabetic rats was significantly higher than non-diabetic rats in both intact and denuded endothelium. In the presence of faclofen, a selective GABA_B receptor blocker, GABA-induced relaxation in intact and denuded endothelium mesenteric beds of STZ diabetic rats was suppressed, but this response in non-diabetic rats was not suppressed. Our results showed that in the presence of L-Arg, a nitric oxide precursor, GABA induced vasorelaxation in both diabetic and non-diabetic vessels.

Conclusion:

From the results of this study, it may be concluded that the vasorelaxatory effect of GABA in diabetic vessel is mediated by the GABA_B receptor and nitric oxide, but it seems that in non-diabetic vessel GABA_B receptor does not play any role in GABA-induced vasorelaxation, but nitric oxide induced GABA relaxation in non-diabetic vessel. Key Words: Gamma amino butyric acid (GABA), Diabetes, GABA_B receptor     

Chronic Aerobic Exercise Decreases Lectin-Like Low Density Lipoprotein (LOX-1) Receptor Expression in Heart of Diabetic Rat

Background:

Overexpression of lectin-like low density lipoprotein (LOX-1) receptor plays an important role in hyperglycemia-induced vascular complications such as atherosclerosis. Based on the beneficial effects of exercise on preventing cardiovascular complications of diabetes, we aimed to examine the protective effects of aerobic exercise on expression of LOX-1 receptor and production of free radicals in the heart of diabetic rats.

Methods:

Four groups of rats were used: (n = 5 per group): sedentary normal, trained normal, sedentary diabetes and trained diabetes. Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg). The exercise protocol was consisted of swimming 30 min/day, 5 days/week for eight weeks. Plasma glucose was evaluated at initiation, weeks 4 and 8 of experiment. At the end of experiment, rats were sacrificed and the heart was removed for determination of nitrate, malondialdehyde, and LOX-1 gene expression.

Results:

In normal non-diabetic rats, the blood glucose level was <150 mg/dl; however, the induction of diabetes resulted in levels more than >400 mg/dl. Gene expression of LOX-1 was increased in the heart of diabetic rats. Exercise reduced the gene expression of this protein in diabetic states without reducing the blood glucose. Finally, swimming exercise decreased the malondialdehyde and nitrate levels in heart tissue both in control and diabetic rats.

Conclusion:

Swimming exercise reduces heart expression of the LOX-1 receptor in accompany with reduction of free radicals production. Since these parameters are important in generation of diabetic complications, swimming exercise is a good candidate for reducing these complications. Key Words: Hyperglycemia, Diabetic complications, LOX-1 receptor, Exercise, Free radicals     

The Role of Biodegradable Engineered Nanofiber Scaffolds Seeded with Hair Follicle Stem Cells for Tissue Engineering

Background

The aim of this study was to fabricate the poly caprolactone (PCL) aligned nanofiber scaffold and to evaluate the survival, adhesion, proliferation, and differentiation of rat hair follicle stem cells (HFSC) in the graft material using electrospun PCL nanofiber scaffold for tissue engineering applications.

Methods

The bulge region of rat whisker was isolated and cultured in DMEM: nutrient mixture F-12 supplemented with epidermal growth factor. The morphological and biological features of cultured bulge cells were observed by light microscopy using immunocytochemistry methods. Electrospinning was used for production of PCL nanofiber scaffolds. Scanning electron microscopy (SEM), 3-(4, 5-di-methylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and histology analysis were used to investigate the cell morphology, viability, attachment and infiltration of the HFSC on the PCL nanofiber scaffolds.

Results

The results of the MTT assay showed cell viability and cell proliferation of the HFSC on PCL nanofiber scaffolds. SEM microscopy images indicated that HFSC are attached, proliferated and spread on PCL nanofiber scaffolds. Also, immunocytochemical analysis showed cell infiltration and cell differentiation on the scaffolds.

Conclusion

The results of this study reveal that PCL nanofiber scaffolds are suitable for cell culture, proliferation, differentiation and attachment. Furthermore, HFSC are attached and proliferated on PCL nanofiber scaffolds.Key Words: Nanofiber, Electrospinning, Stem cells, Tissue engineering     

in vitro Anti-Proliferative Effect of Adiponectin on Human Endometriotic Stromal Cells through AdipoR1 and AdipoR2 Gene Receptor Expression

Background:

Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors.

Methods:

In this experimental study, endometrial biopsies (n=7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48, or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student’s t-test, and P<0.05 was considered statistically significant.

Results:

Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P=0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P<0.05).

Conclusions:

Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.Key Words: Adiponectin, Adiponectin receptors, Endometriosis, Stromal cells     

Comparative Study of Immunological and Structural Properties of Two Recombinant Vaccine Candidates against Botulinum Neurotoxin Type E

Background

Recently, botulinum neurotoxin (BoNT)-derived recombinant proteins have been suggested as potential botulism vaccines. Here, with concentrating on BoNT type E (BoNT/E), we studied two of these binding domain-based recombinant proteins: a multivalent chimer protein, which is composed of BoNT serotypes A, B and E binding subdomains, and a monovalent recombinant protein, which contains 93 amino acid residues from recombinant C-terminal heavy chain of BoNT/E (rBoNT/E-HCC). Both proteins have an identical region (48 aa) that contains one of the most important BoNT/E epitopes (YLTHMRD sequence).

Methods

The recombinant protein efficiency in antibody production, their structural differences, and their BoNT/E-epitope location were compared by using ELISA, circular dichroism, computational modeling, and hydrophobicity predictions.

Results

Immunological studies indicated that the antibody yield against rBoNT/E-HCC was higher than chimer protein. Cross ELISA confirmed that the antibodies against the chimer protein recognized rBoNT/E-HCC more efficiently. However, both antibody groups (anti-chimer and anti-rBoNT/E-HCC antibodies) were able to recognize other proteins. Structural studies with circular dichroism showed that chimer proteins have slightly more secondary structures than rBoNT/E-HCC.

Conclusion

The immunological results suggested that the above-mentioned identical region in rBoNT/E-HCC is more exposed. Circular dichroism, computational protein modeling and hydrophobicity predictions indicated a more exposed location for the identical region in rBoNT/E-HCC than the chimer protein, which is strongly in agreement with immunological results. Iran. Biomed. Key Words: Botulinum neurotoxin type E (BoNT/E), Cross ELISA, circular dichroism, Computational modeling, recombinant vaccine-candidates     

Quantitative Assessment of Proliferative Effects of Oral Vanadium on Pancreatic Islet Volumes and Beta?Cell Numbers of Diabetic Rats

Background:

Oral vanadyl sulfate (vanadium) induces normoglycemia, proliferates beta cells and prevents pancreatic islet atrophy in streptozotocin-induced diabetic rats. Soteriological method is used to quantitate the proliferative effects of vanadium on beta-cell numbers and islet volumes of normal and diabetic rats.

Methods:

Adult male Sprague-Dawley rats were made diabetic with intravenous streptozotocin injection (40 mg/kg). Normal and diabetic rats were divided into four groups. While control normal and diabetic (CD) groups used water, vanadium-treated normal (VTN) and diabetic (VTD) groups used solutions containing vanadyl sulfate (0.5-1 mg/mL, VOSO₄+5H₂O). Tail blood samples were used to measure blood glucose (BG) and plasma insulin. Two months after treatment, rats were sacrificed, pancreata prepared, and stereology method was used to quantitatively evaluate total beta cell numbers (TBCN) and total islet volumes (TISVOL).

Results:

Normoglycemia persisted in VTN with significantly decreased plasma insulin (0.190.08 vs. 0.970.27 ng/dL, P<0.002). The respective high BG (53249 vs. 14446 mg/dL, P<0.0001) and reduced plasma insulin (0.260.15 vs. 0.540.19 ng/dL, P<0.002) seen in CD were reversed in VTD during vanadium treatment or withdrawal. While the induction of diabetes, compared to their control, significantly decreased TISVOL (1.90.2 vs. 3.030.6 mm³, P<0.003) and TBCN (0.990.1 vs. 3.20.2 x 10⁶, P<0.003), vanadium treatment significantly increased TISVOL (2.90.8 and 4.071.0 mm³, P<0.003) and TBCN (1.50.3 and 3.80.6 x 10⁶, P<0.03).

Conclusion:

Two-month oral vanadium therapy in STZ-diabetic rats ameliorated hyperglycemia by partially restoring plasma insulin. This action was through proliferative actions of vanadium in preventing islet atrophy by increasing beta-cell numbers.Key Words: Vanadium, Pancreas, Islet volumes, Rats     

Prevalence of Class 1 Integrons and Extended Spectrum Beta Lactamases among Multi-Drug Resistant Escherichia coli Isolates from North of Iran

Background:

Extended spectrum beta lactamases (ESBLs) are an important cause of transferable multidrug resistance (MDR) in gram-negative bacteria. The most described ESBL genes are generally found within integron-like structures as mobile genetic elements. The aim of this study was to identify the accompanying of class 1 integrons and ESBLs in the MDR E. coli isolates.

Methods:

Susceptibility to antimicrobial agents was determined for 33 E. coli strains by the disk diffusion method. Double-disk synergy test was applied for screening ESBL. To identify the strains carrying integrons, the conserved regions of integron-encoded integrase gene intI1 were amplified. For detection of gene cassettes, 5′CS and 3′CS primers were used.

Results:

All E. coli isolates were identified as multi-drug resistant. More than 50% of the isolates were resistant to tetracycline, cephalothin, cefuroxime, amoxicillin-clavulanic acid, and third generation cephalosporines. Nearly all of the isolates displayed sensitivity to piperacillin. There was a significant correlation between production of ESBL and resistance to all antibiotics except for ciprofloxacin and piperacillin (P < 0.01). Thirty two MDR strains (97%) included class 1 integron, and some isolates that included integrons were similar in the size of gene cassettes. The isolates were different in the resistance profiles; however, some others had similar resistance profiles. Of eight ESBL positive isolates, seven (87.5%) carried class 1 integrons.

Conclusion:

Class 1 integrons were frequent in MDR and also ESBL-producing E. coli isolates. High prevalence of class 1 integrons confirms that integron-mediated antimicrobial gene cassettes are important in E. coli resistance profile. Key Words: Antibiotic, Integrons, Escherichia coli     

Particular Distribution of Enterobacter cloacae Strains Isolated from Urinary Tract Infection within Clonal Complexes

Background:

Based on biochemical properties, Enterobacter cloacae represents a large complex of at least 13 variant species, subspecies, and genotypes that progressively identified as the most species causing hospital-acquired infections. The aim of this study was to determine the relevance between phylogenetically related strains within the E. cloacae complex and the frequency of urinary tract infection caused by them.

Methods:

A 268-bp fragment was obtained from hsp60 gene for 50 clinical E. cloacae isolates from urine cultures of inpatients that admitted to six hospitals in Tehran, Iran during December 2012 to November 2013. The 107 nucleotide sequences were analyzed and the evolutionary distances of sequences were computed and neighbor-joining tree was calculated.

Results:

It showed that all of the genetic clusters have not an equal involvement in pathogenesis of urinary tract infections. Three superior clusters were found, together representing more than two third (80%) of the isolates (cluster VI with 25 members; clusters III and VIII with 9 and 6 members, respectively) and some genetic clusters were absent (IV, X, XII, and xiii), some of which are supposed to be associated with plants and no human infection has been reported.

Conclusions:

This study, for the first time, reports the unequal contribution of E. cloacae complex subspecies and clusters in urinary tract infections in Iran and together with studies from other countries suggest that the subspecies of E.hormaechei subsp. Oharae is the most prevalent E. cloacae complex subspecies regardless of country under study. Key Words: Enterobacter cloacae complex, Urinary tract infection, hsp60     

Chitosan-Based Intranasal Vaccine against Escherichia coli O157:H7

Background:

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an infectious zoonotic pathogen causing human infections. These infections, in some cases, can lead to hemolytic uremic syndrome and its life-threatening complications and even death worldwide. The first intimate bacterial adhesion, intimin (I), with its own receptor translocated intimin receptor (Tir) and E. coli secreted protein A, acting as Tir conduit, are highly immunogenic proteins for vaccine development against E. coli O157:H7.

Methods:

A chimeric trivalent recombinant protein was previously found to be a suitable strategy for developing vaccines against E. coli O157:H7. In this study, the recombinant EIT (rEIT) was used to design a protective EHEC nasal nanovaccine. Chitosan and its water-soluble derivative, trimethylated chitosan (TMC), as muco-adhesive biopolymers, are good candidates for preparation of nanovaccines.  Using the electrospraying technique, as a novel method, we could obtain particles of rEIT loaded with chitosan and TMC on a nanometer scale. Mice were immunized with intranasal administration or intrapretoneal injection of rEIT.

Results:

The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. Only nasal administration of chitosan electrospray and TMC formulation produced significant secretion IgA. Intranasal administration of nanovaccine reduced the duration of bacterial fecal shedding on mice challenged with E. coli O157:H7.

Conclusion:

Since development of mucosal vaccines for the prevention of infectious diseases requires efficient antigen delivery; therefore, this research could be a new strategy for developing vaccine against E. coli O157:H7.Key Words: EnterohemorrhagicEscherichia coli, Nanoparticles, Intranasal vaccination     

Association Study of rs1333040 and rs1004638 Polymorphisms in the 9p21 Locus with Coronary Artery Disease in Southwest of Iran

Background:

Coronary artery disease (CAD) is a multifactorial and heterogenic disease. Recently, genome-wide association studies have reported that rs1333040 (C/T) and rs1004638 (A/T) single nucleotide polymorphisms (SNPs) in the 9p21 locus have very strong association with CAD. This study aimed to examine these associations in Southwest of Iran.

Methods:

Blood samples were collected from 200 CAD patients and 110 healthy individuals with no CAD. The association of two SNPs with CAD was evaluated by PCR and restriction fragment length polymorphism.

Results:

Chi-square test showed no association between rs1333040 SNP and CAD (X²: 4.66, df: 2, P=0.09). Also, there was no association between rs1004638 SNP and CAD (X²: 0.27, df: 2, P=0.88).

Conclusion:

No association was observed between rs1333040 and rs1004638 SNPs in the 9P21 region and CAD in Southwest of Iran. Key Words: Coronary artery disease, Single nucleotide polymorphisms, Genetic association study, Iran     

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

Background:

Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.

Results:

The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.

Conclusion:

Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)     

Protective Role of Oleuropein against Acute Deltamethrin-Induced Neurotoxicity in Rat Brain

Background:

Deltamethrin (DM) is a synthetic pyrethroid insecticide that can elicit neurotoxicity, leading to apoptosis. There is accumulating evidence that oleuropein (OE) has anti-apoptotic effect. The purpose of this study was to determine the anti-apoptotic effect of OE pretreatment in the neuronal cells of cerebral cortex.

Methods:

Rats were randomly divided into four groups each containing five rats: DM-treated group (12.5 mg/kg, a single dose), OE-treated group (20 mg/kg per day), DM + OE-treated group, and vehicle group. Sections of the brain were obtained 24 hours after DM injection and studied for histopathological and immunohistochemistry assessment.

Results:

The histopathological assessments showed lesser characteristics of neural degeneration in DM + OE group compared with DM group. Greater Bcl-2 and attenuated Bax expression could be detected in the DM + OE treated-mice compared with DM group.

Conclusion:

The results suggested that DM-induced neurotoxicity can be subsided by OE.Key Words: Deltamethrin, Oleuropein, Apoptosis     

Molecular Genetic Analysis of the Variable Number of Tandem-Repeat Alleles at the Phenylalanine Hydroxylase Gene in Iranian Azeri Turkish Population

Background:

The variable numbers of tandem-repeat (VNTR) alleles at the phenylalanine hydroxylase (PAH) gene have been used in carrier detection and prenatal diagnosis in phenylketonuria families. This study was carried out to analyze VNTR alleles at the PAH gene in Iranian Azeri Turkish population.

Methods:

In this study, 200 alleles from general population were studied by PCR.

Results:

The frequencies of VNTR alleles were 45%, 46%, 2%, 3%, 1%, and 3% in studied group regarding 3, 8, 9, 11, 12, and 13 repeat copies, respectively. Statistically significant differences were not found between expected and observed frequencies of VNTR genotypes (P > 0.05).

Conclusions:

VNTR alleles with three and eight repeats were frequent, and the VNTR alleles with 13 repeats showed 3% frequency in the tested group. This study is the first report on tested population genetic structure using VNTR alleles at the PAH gene. Key Words: Phenylalanine hydroxylase, Population genetics, Variable numbers of tandem-repeat     

Association of Lecithin Cholesterol Acyltransferase rs5923 Polymorphism in Iranian Individuals with Extremely Low High-Density Lipoprotein Cholesterol: Tehran Lipid and Glucose Study

Background:

The serum concentration of high-density lipoprotein cholesterol (HDL-C) is one of the important heritable risk factors for cardiovascular disease and is a target for therapeutic intervention. In this study, we aimed to evaluate the effects of lecithin cholesterol acyltransferase (LCAT) gene polymorphism rs5923 on LCAT enzyme activity and serum HDL-C concentration.

Methods:

The study population was selected from consecutive individuals with HDL-C ≤ 5^th percentile (n = 73) and extremely high HDL-C ≥ 95^th percentile (n = 57) who had participated in the Tehran Lipid and Glucose Study. The rs5923 polymorphism was genotyped using direct sequencing. LCAT activity was measured by fluorometric assay kit, and lipid concentrations were measured using the enzymatic colorimetric method.

Results:

The genotype frequencies were significantly different between the high HDL-C group (CC 94.7%, CT 5.3%) and the low HDL-C group (CC 83.6%, CT 16.4%) (P = 0.048). The T-allele frequencies in subjects with low and high HDL-C were 0.082 and 0.026, respectively (P = 0.16). The association of the single-nucleotide polymorphism rs5923 with low HDL-C was not statistically significant after adjustment for age, sex, and BMI (odd ratio = 2.65, 95% confidence interval = 0.32-21.5, P = 0.36, regression logistic analysis). Also, the effects of LCAT enzyme activity did not depend on the HDL-C level (P = 0.24).

Conclusion:

rs5923 polymorphism is not associated with low HDL-C levels in Iranian population. Key Words: Polymorphism, Single nucleotide, Lipoproteins     

Heat Shock Proteins Enriched-Promastigotes of Leishmania major Inducing Th2 Immune Response in BALB/c Mice

Background

Heat shock proteins (HSP) are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen (SLA) was determined.

Methods

BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 10⁶ stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks.

Results

No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-γ, IL-4, TGF-β, IgG1 and IgG2b were increased in both groups, IFN-γ was significantly higher in SLA group and IgG2a in HSP-enriched SLA.

Conclusion

These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection. Key Words: Leishmaniasis, Heat shock proteins (HSP), Adjuvant     

A Novel Mutation in the Aprataxin (APTX) Gene in an Iranian Individual Suffering Early-Onset Ataxia with Oculomotor Apraxia Type 1(AOA1) Disease

Background

Ataxia with oculomotor apraxia type 1 (AOA1) shows early onset with autosomal recessive inheritance and is caused by a mutation in the aprataxin (APTX) gene encoding for the APTX protein.

Methods

In this study, a 7-year-old girl born of a first-cousin consanguineous marriage was described with early-onset progressive ataxia and AOA, with increased cholesterol concentration and decreased albumin concentration in serum. PCR and direct DNA sequencing was performed after DNA extraction.

Results

Sequencing analysis revealed a novel homozygous deletion in c.643 and A>T single nucleotide polymorphism in c.641 in exon 6 of the APTX gene [ENST00000379825].

Conclusion

It seems that this region of exon 6 is probably a hot spot; however, no deletions have been reported in exon 6 yet. Key Words: Ataxia oculomotor apraxia 1 (AOA1), aprataxin (APTX), Iranian     

Effects of Peptone Supplementation in Different Culture Media on Growth,Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles

Background:

The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood.

Methods:

In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes.

Results:

In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium.

Conclusion:

The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.Key Words: CHO cells, Culture media, Peptones, Recombinant proteins     

Efficient Process Development of Recombinant Human Granulocyte Colony-Stimulating Factor (rh-GCSF) Production in Escherichia coli

Background:

The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients.

Methods:

An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h^-1 at the beginning of feeding to 0.4 h^-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography.

Results:

Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l^-1 and 32.1 g.l^-1. Also, the final specific yield (Y_P/X) and total productivity of rh-GCSF were obtained 254 mg.g^-1 DCW and 1.83 g.l^-1.h^-1, respectively. According to the available data, this is one of the highest Y_P/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen® and PD-Grastim showed that all proteins have a similar secondary structure.

Conclusion:

Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity. Key Words: Escherichia coli, Granulocyte colony-stimulating factor (GCSF), Process development     

Protective Effects of Restricted Diet and Antioxidants on Testis Tissue in Rats Fed with High-Fat Diet

Background:

A high-fat diet (HFD) promotes the oxidative stress formation, which in turn has hazardous effects on reproductive system and fertility. The present study examines the potential positive effects of a restricted high-fat diet (RHFD) and antioxidants consumption on sperm parameters and testis tissue in rats.

Methods:

Male rats (n = 48) were divided into four groups (12 in each group): control group (Cont), HFD group, RHFD, and RHFD with astaxanthin and vitamins E and C group (RHFDA). After 12 weeks, serum analysis and sperm parameters study were performed. Sections of fixed testes were stained with Hematoxilin and Eosin to study the histological changes. A one-way ANOVA was used to compare the data.

Results:

HFD fed animals presented significant increase in weight load and serum low density lipoprotein (LDL-C) levels (P < 0.05). The sperm count in RHFD was lower than three other groups (P < 0.05) and sperm motility of RHFDA group was significantly higher than HFD and RHFD groups (P < 0.05). The histological study was showed a significant increase in spermatogonium number in RHFDA compared to three other groups (P < 0.05). The number of spermatocyte I and spermatid in RHFD was significantly (P < 0.05) lower than Cont and HFD groups.

Conclusion:

HFD and obesity can affect sperm parameters and spermatogenesis and antioxidants consumption may improve their quality. Although the RHFD is a benefit way in weight loss and decrease of LDL-C of serum, but it is suggested that is not effective on sperm quality improvement.Key Words: Spermatogenesis, high-fat Diet, Restricted fat diet, Antioxidants, Astaxanthin