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)     

Photochemical Formation of Hydroxyl Radicals in Red Soil-Polluted Seawater on the North of Okinawa Island, Japan

Land development has caused runoff of red soil into the ocean on the north side of Okinawa Island, Japan. In an attempt to clarify the impacts of this “red soil pollution” on the oxidizing power of seawater, we studied the formation of hydroxyl radical (?OH), the most potent oxidant in the environment, in red soil-polluted waters using a 313-nm monochromatic light. ?OH was photochemically formed in the red soil-polluted water samples, and the formation rates of ?OH decreased as salinity increased, i.e., as red soil-polluted river water gets mixed with seawater. The photo-formation rates of ?OH showed good correlations with dissolved Fe concentrations (R ²?=?0.96) and [NO₂ ^?]?+?[NO₃ ^?] concentrations (R ²?=?0.87), while a negative and weak correlation was found with dissolved organic carbon concentrations (R?=??0.78). Theoretical calculation showed that direct photolysis of NO₃ ^?, Fe(OH)²⁺, and hydrogen peroxide all together accounted for less than 10% of the observed ?OH formation in the red soil-polluted waters. Comparison between filtered and unfiltered samples showed that red soil particles were not the main sources of ?OH, and the photolysis of NO₂ ^? could account for at most 78% of the observed ?OH formation rates. We found that the Fenton’s reaction (a reaction between Fe(II) and H₂O₂) could possibly account for the observed formation of ?OH in the red soil-polluted waters.     

Evaluation of lung pathology in Dirofilaria immitis-experimentally infected dogs treated with doxycycline or a combination of doxycycline and ivermectin before administration of melarsomine dihydrochloride

Adulticide therapy in heartworm (Dirofilaria immitis)-infected dogs can lead to thromboembolism, which can seriously compromise post-treatment health status. Lung pathology following adulticide therapy was evaluated in three groups of experimentally infected dogs. Group 1 was treated with doxycycline at 20 mg/kg per os once daily for 30 days post infection followed by an intramuscular injection of melarsomine dihydrochloride (2.5 mg/kg) at Week 12, followed 1 month later by two injections 24 h apart. Group 2 was treated as described for Group 1, with the addition of ivermectin at 6 mcg/kg given monthly per os for 24 weeks post-infection. Group 3 received melarsomine alone, as described above. All dogs were necropsied at Week 24 and lung pathology was evaluated. Lesion criteria included perivascular inflammation and endothelial proliferation. Lesions were scored by two independent pathologists who were blinded as to treatment. Results indicate that doxycycline treatment alone or combined with ivermectin had lower lesion scores than lungs from dogs who had received melarsomine alone. Dogs that received the combined doxycycline/ivermectin protocol and treated with adulticide showed less severe arterial lesions and the virtual absence of thrombi.     

Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities

Taking into account the recalcitrance of date palm (Phoenix dactylifera L.) to tissue culture, establishment and proliferation of embryogenic suspension cultures in two date palm cultivars, namely Boufeggouss (BFG) and Bouskri (BSK) was implemented using liquid medium with different concentrations of the 6-benzylaminopurine (BAP) (0.3, 0.4, 0.5 mg/l) and various subculturing times (7 days, 15 days and 20 days). Total phenols and peroxidase activities have been determined and oxidative browning process checked in different cultures. The liquid medium with 0.3 mg/l BAP allowed cell suspension cultures to produce the greater globular embryos and the highest number of somatic embryos in comparison with the other treatment for BFG and BSK date palm cultivars. The low rate of culture transfers (every 15 or 20 days) tended to increase the phenolic contents and peroxidase activities in plant tissue leading to an enhancement of tissues/cells browning and then a decrease in the proliferation of embryogenic cells. The transfer of cultures on fresh medium every 7 days allowed a substantial reduction of tissue/cell oxidative browning, which is related to reduction of phenolic compounds and decrease in peroxidase activities with a resultant increase in the proliferation of embryogenic cells. This result suggests that oxidative browning is mainly peroxidase-based in date palm suspension cultures. Relationships between low concentrations of BAP as cytokinin, high growth of embryogenic cells, low contents of phenolics, decrease of peroxidase activities and oxidative browning process, are discussed, in order to optimize date palm multiplication through somatic embryogenesis and solve recalcitrance of many date palm cultivars to tissue culture.     

Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein

In mammals, hair cell loss causes irreversible hearing and balance impairment because hair cells are terminally differentiated and do not regenerate spontaneously. By profiling gene expression in developing mouse vestibular organs, we identified the retinoblastoma protein (pRb) as a candidate regulator of cell cycle exit in hair cells. Differentiated and functional mouse hair cells with a targeted deletion of Rb1 undergo mitosis, divide, and cycle, yet continue to become highly differentiated and functional. Moreover, acute loss of Rb1 in postnatal hair cells caused cell cycle reentry. Manipulation of the pRb pathway may ultimately lead to mammalian hair cell regeneration.     

Effects of dietary protein level on spawning performance of Nile tilapia (Oreochromis niloticus) broodstock reared at different water salinities

Effects of dietary protein level and water salinity on spawning performance of Nile tilapia broodstock and growth of their larvae were studied. Four isocaloric (400 kcal/100 g) diets containing 25%, 30%, 35% and 40% crude protein were prepared. The diets were fed to broodfish (25.7 g) reared at three water salinities (0‰, 7‰ and 14‰) at a female/male ratio of 3:1, to satiation twice a day for 195 days. The size at first maturation increased with increasing dietary protein at all salinities. At 25% and 30% protein levels, broodstock reared at 0‰ reached their sexual maturity at bigger sizes than those reared at 7‰ and 14‰. At 0‰, spawning intervals were not significantly affected by dietary protein levels. At 7‰ and 14‰, spawning intervals significantly decreased with increasing dietary protein level. Spawning frequency and number of eggs per spawn were increased with increasing dietary protein level. The total number of spawnings per female and absolute fecundity were better in fish fed 40% protein in freshwater than at 7‰ and 14‰ salinity. The relationship of dietary protein and water salinity on egg size was significant, but showed irregular patterns. The chemical composition of broodstock muscles, eggs and fry were not significantly affected by dietary protein and water salinity, except for body water and crude protein of broodstock which were significantly affected; but showed irregular trends. At each water salinity, egg hatchability was linearly increased with increasing dietary protein level. Eggs produced from broodstock fed 25% protein at 7‰ and 14‰ needed more time for hatching and yolk-sac absorption and resulted in poorer larval weight than those reared in freshwater. Fry growth was improved with increasing protein level at all salinities. This result revealed that 40% dietary protein is required for optimum spawning performance of Nile tilapia reared at 0‰, 7‰ and 14‰ salinity. It also indicated that spawning performance and larval growth were better in freshwater than at 7‰ and 14‰.     

Growth, Survival and Feed Conversion Rates of Sea Bream (Sparus aurata) Cultured in Earthen Brackish Water Ponds Fed Different Feed Types

Sparus aurata were cultured during an 8-month period in brackish water (salinity about 25 ppt) in an extensive culture system comprising eight earthen ponds, each with a water surface of 2.1 ha. Initial mean wet weight of fish in all ponds varied from 13.6 ± 1.9 to 19.2 ± 2.6 g/fish. The eight ponds were randomly allocated one of four experimental treatments (two ponds/treatment). In the first treatment, ponds were fertilized monthly with 100 kg urea and 50 kg triple super phosphate. The other treatments (2–4) were fed a locally produced tilapia pellet feed containing 25% crude protein made using different processes. Fish in the second treatment were fed tilapia feed pelleted by compressing machine, whereas in treatments 3 and 4 the pellets were produced by extruder machine (Wenger). Pellets in treatment 3 were floating and in the fourth were semi-sinking. Fish were fed pellets twice daily at 6% of their biomass. The mean final body weight for each treatment respectively was 104.6, 118.9, 156.8 and 158 g. Specific growth rate (SGR) of 0.8, 0.79, 0.99 and 0.95%/day, were obtained in ponds using only inorganic fertilizer, compressed sinking pellets, extruded floating pellets and extruded semi-sinking pellets, respectively. Feed conversion ratios (FCR) for treatments with the extruded tilapia pellets were 2.2 and 2.6 g feed/g gain, which were significantly (P < 0.05) better than treatments with compressed pellets (3.2 g feed/g gain). Production/ha/year were 1389, 1358, 945 and 682 kg fish for the groups fed with extruded floating, extruded semi-sinking, compressed and natural food, respectively. Under the present experimental circumstances, Sparus aurata fed extruded floating tilapia pellets (25% crude protein and 2,600 kcal/g), showed the best productive performance.     

Effect of different concentrations of ginger root powder and its essential oil on growth performance,serum metabolites and antioxidant status in broiler chicks under heat stress

1. This study was carried out to evaluate the impact of ginger (Zingiber officinale) feed supplementation on growth performance, antioxidant status, carcass characteristics and blood parameters in broiler chicks under conditions of heat stress (32 ± 2ºC for 8 h per d).

2. A total of 336 d-old male broiler chicks (Cobb-500) were randomly assigned to one of 6 dietary groups representing: basal diet with no supplement as control, basal diet containing 100 mg/kg vitamin E as positive control, basal diets containing either 7.5 or 15 g/kg of ginger root powder, and diets containing 75 or 150 mg/kg of ginger essential oil.

3. The results indicated that at 22 d of age, the group receiving 7.5 g/kg of ginger root powder experienced significantly increased body weight (BW) and body weight gain (BWG) compared to the control group. There were no significant difference among the diet groups regarding BW, BWG, feed intake (FI) or feed conversion ratio (FCR) at 42 and 49 d of age.

4. The inclusion of powder and essential oil of ginger in broiler diets did not affect carcass characteristics and blood parameters of the chickens. However, in the group receiving 150 mg/kg ginger essential oil, the total superoxide dismutase (TSOD) activity in liver increased compared to the control group. Malondialdehyde (MDA) concentrations in liver also decreased in the groups receiving ginger powder and essential oil compared to that in the control group. There were no significant difference between experimental groups regarding glutathione peroxidise (Gpx), TSOD and catalase (CAT) enzymes in red blood cells. All dietary groups increased total antioxidant capacity (TAC) and decreased MDA concentration in serum compared to the control group.

5. The results of this study suggest that ginger powder and essential oils may be a suitable replacement for synthetic antioxidants in broiler diets. Results also suggest that ginger powder might be better than extracted essential oil for improving antioxidant status in broilers.     

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.