Novel mutation in the EXT-1 gene in an Iranian family affected with hereditary multiple exostoses

Identification of casual mutations in Hereditary Multiple Exostoses (HME) is important because of similar conditions in which multiple exostoses occur. Therefore mutation analysis can help to confirm the clinical diagnosis and to improve the management of therapy. HME is an inherited disorder of bone growth. HME can be referred to by various names such as Heredity Multiple Exostoses, Hereditary Multiple Osteochondromata, Multiple Carthaginous Exostoses, etc. People who have HME grow exostoses, or bony bumps, on their bones which can vary in size, location and number depending on the individual. HME is inherited in an autosomal dominant manner with an estimated prevalence of 1/50,000 in western countries. At least three loci (EXT1, EXT2 and EXT3) thought to be involved in this skeletal disease. Approximately 90% of affected families possess mutations in the coding regions of EXT1 and EXT2 genes and the majority of these mutations cause loss of function. EXT1 and EXT2 genes encode related members of a putative tumor suppressor family. In this first report from Iran we identified a frame shift mutation (1100-1101 insA) in exon 3 of EXT1 gene in a family being suspicious of HME. This mutation leads to a premature stop codon and previously not described. Additionally, we have found an unreported silent mutation in the exon six of EXT1 gene with uncertain significance.     

Screening of sugar beet tissue culture clones for resistance to rhizomania disease

In this study, sugar beet tissue culture clones were used to screen rhizomania resistant genotypes. At first, explants derived from shoot tips of sugar beet seedlings were transferred to shoot tip elongation media after surface sterilization. Then, the grown shoots were transferred to media containing various hormonal combinations NAA, BA, IBA and GA3 for multiplication, growth and rooting. Later, the clones were transferred to soil-peatmoss mixture were adapted to greenhouse conditions. For screening clones against rhizomania, the genotypes of adapted clones were selected and inoculated to rhizomania-infested soil. This experiment was in a randomized complete block design with three replicates (three inoculation times) in greenhouse. Adapted plants were transferred to the soil containing rhizomania virus. All infested soils were diluted 3 to 7 with sand. After two months, infested plants were examined by DAS-ELISA test also optical densities of the samples were analyzed by SAS program. Significant differences among genotypes and blocks were observed. Genotypes were classified to few groups (ranked from completely susceptible to completely resistant). The difference between blocks was because of difference of inoculation time temperature. Use of clones of each genotype caused an increase in selection accuracy of resistant genotypes. By use of this method, chance of escaping from inoculation factor decrease and researchers can determine to be resistance of plants with high level of confidence and apply in breeding programs.     

AT1 receptors activation enhances the expression of MMP-2, MMP-13 and VEGF but not MMP-9 in B16F10 melanoma cells

Melanoma is one of the most aggressive cancers of all solid tumors. The effect of angiotensin II on expression of three Matrix Metalloproteinases (MMPs) and Vascular Endothelial Growth Factor (VEGF) in B16F10 melanoma cells was evaluated. Also the blocking effect of losartan on angiotensin II induced effects was assessed. B16F10 murine melanoma cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 24 h prior to experiment the serum free medium was used. Angiotensin II (0 M, 10(-10) M, 10(-9) M or 10(-8) M) alone or in combination with Losartan (10(-6) M) in RPMI-1640 replaced the medium for experiments. After the incubation time (0, 1, 2, 6 and 12 h) cells were harvested using 0.05% (w/v) Trypsin and then recovered by centrifugation. The expression of MMP-2, MMP-13, MMP-9 and VEGF in B16F10 cell lysate was assessed by immunoblotting. Angiotensin II significantly enhanced the expression of MMP-2, MMP-13 and VEGF by concentrations as low as 0.1 nM. But angiotensin II could not stimulate any significant increase in MMP-9 expression by angiotensin II in B16F10 cells. Losartan abolished the enhancing effect of every concentration of angiotensin II on MMP-2, MMP-13 and VEGF expression completely and in all incubation times. As a result, angiotensin II through activation of AT1 receptors can stimulate the expression of MMP-2, MMP-13 and VEGF in B16F10 melanoma cells. This is an important conclusion because of the importance of these factors in melanoma invasiveness and the possible important role that angiotensin receptor blockers may play as cancer medications.     

Bioethanol production from sweet sorghum bagasse by Mucor hiemalis

The present work deals with production of ethanol from sweet sorghum bagasse by a zygomycetes fungus Mucor hiemalis. The bagasse was treated with phosphoric acid and sodium hydroxide, with or without ultrasonication, prior to enzymatic hydrolysis by commercial cellulase and β-glucosidase enzymes. The phosphoric acid pretreatment was performed at 50 °C for 30 min, while the alkali treatment performed with 12% NaOH at 0 °C for 3 h. The pretreatments resulted in improving the subsequent enzymatic hydrolysis to 79-92% of the theoretical yield. The best hydrolysis performance was obtained after pretreatment by NaOH assisted with ultrasonication. The fungus showed promising results in fermentation of the hydrolyzates. In the best case, the hydrolyzate of NaOH-ultrasound pretreated bagasse followed by 24 h fermentation resulted in about 81% of the corresponding theoretical ethanol yield. Furthermore, the highest volumetric ethanol productivity was observed in the hydrolyzates of NaOH pretreated bagasse, especially after ultrasonication in pretreatment stage.     

Prophylactic and Therapeutic Effects of Oleuropein on Reperfusion-Induced Arrhythmia in Anesthetized Rat

Background:

This study was conducted to reveal that whether i.v. injection of oleuropein, the most potent polyphenolic antioxidant in olive leaf, has any effect on the magnitude of reperfusion arrhythmia in anesthetized rats or not.

Methods:

Eighty male Wistar rats were divided into 8 groups of 10 each: groups 1 and 5 were assigned as the prophylactic and treatment control groups, groups 2 and 6 as the prophylactic and treatment groups with lidocaine (10 mg/kg), groups 3 and 4 as the prophylactic groups with 10 and 50 mg/kg oleuropein (i.v.), and groups 7 and 8 as the treatment groups with 10 and 50 mg/kg oleuropein (i.v.), respectively. Reperfusion injury was induced by 5-min regional ischemia and 15-min reperfusion of left anterior descending coronary artery. Heart rate, blood pressure, and electrocardiogram were monitored throughout the procedure.

Results:

blood pressure was significantly decreased by infusion of 50 mg/kg oleuropein in groups 4 and 8, but unlike the lidocaine as a standard anti-arrhythmic drug in groups 2 and 5 had not significant effect on heart rate. The onset of arrhythmia in groups received oleuropein (groups 3, 4, 7, and 8) was significantly delayed. The mortality rate due to irreversible ventricular fibrillation was also significantly reduced in groups 3, 4, 7, and 8. The effect of lidocaine in groups 2 and 5 was more potent than that in oleuropein group.

Conclusion:

These findings indicate that i.v. injection of oleuropein possibly through its antioxidant activity reduces the magnitude of reperfusion-induced arrhythmia.Key Words: Oleuropein, Arrhythmia, Reperfusion, Rats     

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)     

Effects of deoxynivalenol exposure time and contamination levels on rainbow trout

The trend toward using plant‐based ingredients in aquafeeds is set to intensify; however, mycotoxin contamination might be a challenge. Two diets, with deoxynivalenol (DON) levels of 1,166 μg/kg (1.1 DON) and 2,745 μg/kg (2.7 DON), were prepared for short‐term DON exposure (50 days). A third diet with a low DON level of 367 μg/kg (0.3 DON) was prepared for long‐term DON exposure (168 days). Ingestion of DON by trout during both short‐term/high‐dosage exposure (50 days; 1,166 μg/kg and 2,700 μg/kg DON) and long‐term/low‐dosage exposure (168 days; 367 μg/kg DON) impacted growth performance and, to a lesser extent, liver enzyme parameters (2.7 DON). Histopathology showed mild to moderate changes in the liver but not in the other sampled tissues (intestine and kidney). Despite these effects, short‐term exposure of rainbow trout to high doses of DON did not result in increased susceptibility to Yersinia ruckeri. In both the short‐ and long‐term studies, the effects of DON showed a high interindividual variability. The present study confirms that subclinical levels of mycotoxins affect rainbow trout. The effects of such low mycotoxin levels could be masked by other production challenges while still negatively affecting productivity.     

Integrated mite management in apples in Israel: Augmentation of a beneficial mite and sensitivity of tetranychid and phytoseiid mites to pesticides

Augmentative releases of the phytoseiid miteTyphlodromus athiasae were evaluated during summer 1987 in an apple orchard designated to be maintained under an IPM (integrated pest management) regime. Evaluation for establishment and recovery 2 weeks after each release showed complete absence of the mite and high population density of the injurious tetranychid mites. It was suspected that pesticides that had been used to control key pests may have been the cause. Laboratory tests with the pesticides used showed that the insect growth regulators triflumuron and fenoxycarb and the fungicide triadimenol caused only slight mortality of the predacious mitesT. athiasae (0-12%) andPhytoseiulus persimilis (6–10%), but a highly significant reduction in fecundity. Four days after treatment a reduction of 94%, 74% and 100% in fecundity ofT. athiasae, and 2 days after treatment a reduction of 78%, 53% and 80% in fecundity ofP. persimilis was brought about by triflumuron, fenoxycarb and triadimenol, respectively. On the other hand, 4 days after treatment there was a 27%, 16% and 9% increase in fecundity of the phytophagous miteTetranychus cinnabarinus caused by triflumuron, fenoxycarb and triadimenol, respectively. Laboratory and field tests were carried out to evaluate 13 other pesticides as to their selectivity toT. athiasae. The compounds azinphos-methyl, penconazole, vamidothion and diflubenzuron were found to be relatively harmless to this beneficial mite and they replaced the materials mentioned above in the coming seasons in several orchards. This caused recovery in high density of the predacious mite, which allowed reduced application of acaricides and the achievement of integrated mite management.     

Effects of Artificial Pollination Using Pollen Suspension Spray on Nut and Kernel Quality of Pistachio Cultivar Owhadi

Yield and nut quality in pistachio are highly dependent on cultural practices especially pollination. This study was conducted to investigate the effects of pollen suspension spray containing 0.01% and 0.02% boric acid combined with 1% (w/v) pollen on nut and kernel quality of pistachio (Pistacia vera L. cv. Owhadi). The experiment was carried out as RCBD with three replications over 2011–12 period. The results showed that application of medium containing agar (A) and 0.01% boric acid (B1) combined with 1% (w/v) pollen decreased fruit set in both years compared to control whereas nut splitting was increased during 2012. Pollen suspension sprays unaffected nut and kernel dimensions in both years but increased kernel proteins and decreased oil during 2011. According to this study, it could be postulated that artificial pollination in pistachio using pollen suspension spray is possible.     

Extensive release of an antigen associated with the sporogonic stages of myxobolus cerebralis (Myxozoa: Myxosporea) is detected by a heterologous antibody raised to Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea)

Monoclonal antibody B4 (mAb B4) was previously developed to the myxozoan parasite Tetracapsuloides bryosalmonae Canning, Curry, Feist, Longshaw et Okamura, 1999, the causative agent of proliferative kidney disease of salmonids, Here we describe the reaction of mAb B4 against Myxobolus cerebralis Hofer, 1903, the parasite that causes 'whirling disease' in salmonids. Tissues examined were collected from experimentally infected rainbow trout Oncorhynchus mykiss (Walbaum) and the aquatic oligochaete Tubifex tubifex (O.F. Müller), the two hosts involved in the life cycle of M. cerebralis. Paraffin sections of infected rainbow trout taken at 4 h and 3, 10, 17 and 54 days post-exposure to infective M. cerebralis actinospores were immunohistochemically stained with mAb B4. Longitudinal sections through infected T. tubifex sampled 120 days post-exposure to M. cerebralis myxospores were also examined using this method. The only phase of the M. cerebralis life cycle that expressed the mAb B4 antigen was during sporogenesis in the salmonid host. The immunohistochemical staining demonstrated that the antigen was released into the tissues surrounding the spore and sporogonic stages of the parasite. The localisation of the antigen was diffuse in the fish, suggesting that the possible effect of M. cerebralis infection is extensive through the head tissues and not limited to areas of cartilage destruction as previously thought.