Immobilization of Urease onto Modified Egg Shell Membrane through Cross Linking

Background:Immobilization is an approach in industry to improve stability and reusability of urease. The efficiency of this technique depends on the type of membrane and the method of stabilization. Methods:The PEI-modified egg shell membrane was used to immobilize urease by absorption and glutaraldehyde cross-linking methods. The membranes were characterized by FTIR and AFM, and Nessler method was applied to measure the kinetic of the immobilized enzymes. Finally, the storage stability (6 °C for 21 days) and reusability (until enzyme activity reached to zero) of the immobilized enzymes were investigated. Results:Based on FTIR, three new peaks were observed in both the absorption- (at 1389.7, 1230.8, and 1074.2 cm^-1) and the cross-linking (at 1615-1690, 1392.7, 1450 cm^-1) immobilized enzymes. The surface roughness of the native membrane was altered after PEI treatment and enzyme immobilization. The optimal pH of cross-linking immobilized enzymes was shifted to a more neutral pH, while it was alkaline in adsorption-immobilized and free enzymes. The reaction time decreased in all immobilized enzymes (100 min for free enzyme vs. 60 and 30 min after immobilizing by adsorption and cross-linking methods, respectively). The optimal temperature for all enzymes was 70 °C and they had a higher K_m and a lower V_max than free enzyme. The stability and reusability of urease were improved by both methods. Conclusion:Our findings propose these approaches as promising ways to enhance the urease efficiency for its applications in industries and medicines. Key Words: Egg shell, Immobilization, Polyethylenimine, Urease     

Induction of Immunogenic Response in BALB/c Mice by Live and Killed Form of Recombinant Lactococcus lactis Displaying EG95 of Echinococcus granulosus

Background:CE is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods:The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results:Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. sIgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion:Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice. Key Words: Echinococcus granulosus, Lactococcus lactis, Immunization, Vaccines     

Hypocholesterolemic effects of dietary soybean vs. casein proteins in a crossed over diets in rat

To investigate the effects of dietary proteins on the level of serum total cholesterol (TC), triglyceride (TG) and high density lipoprotein (HDL), 32 male Wistar rats were randomly divided in control and 3 experimental groups (El, E2 and E3). The feeding regimes of rats were as follow: control, standard diet; E1, a cholesterol free diet containing 20% soybean protein; E2, a cholesterol free diet containing 20% casein and E3, a cholesterol free diet containing 10% soybean protein and 10% casein. The experimental period was 11 weeks but at the end of 7th week the diets of E1 and E2 groups were crossed over for the next 4 weeks. Blood samples were collected weekly, via the ophthalmic sinus and the serum levels TC, TG and HDL were measured. In comparison with control group, the results show that at the end of 7th week TC levels in E1 and E2 groups were significantly (p<0.05) increased while HDL level unchanged and the TC value of E2 was bigger (not significant) than E1. However by crossing over the diets, the TC level was significantly (p<0.05) diminished in E2 while TG value remarkably (p<0.05) increased. These results indicate that soybean protein may insert its hypocholesterolemic effect in hypercholestrolemic condition than in normolipidemic condition.     

Effects of onion on serum uric acid levels and hepatic xanthine dehydrogenase/xanthine oxidase activities in hyperuricemic rats

The aim of this study was to investigate the effects of onion on serum uric acid levels and hepatic Xanthine Dehydrogenase/Xanthine Oxidase activities in normal and hyperuricemic rats. Hyperuricemia was induced by intraperitoneal injection of 250 mg kg(-1) potassium oxonate in rats. Oral administration of onion at 3.5 and 7.0 mg kg(-1) day(-1) for 7 days was able to reduce serum uric acid levels in hyperuricemic rats with no significant effects on the level of this compound in the normal animals. In addition, onion when tested in vivo on rat liver homogeneities elicited significant inhibitory actions on the Xanthine Dehydrogenase (XDH) and Xanthine Oxidase (XO) activities. This effect resulted less potent than that of allopurinol. However, the hypouricemic effect observed in the experimental animal did not seem to parallel the change in XDH and XO activities, implying that the onion might be acting via other mechanisms apart from simple inhibition of enzyme activities. Such hypouricemic action and enzyme inhibitory activity of onion makes it a possible alternative for allopurinol, or at least in combination therapy to minimize the side-effects of allopurinol, in particular in long-term application.     

Comparative Assessment of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Cystatin C as Early Biomarkers for Early Detection of Renal Failure in Patients with Hypertension

Background:

Hypertension is one the most common causes of chronic kidney disease (CKD). One of the major concerns in hypertensive patients is early detection of renal disorders. In the past, serum creatinine (Scr) concentration was used as a marker of kidney function, but it proffers a late reflection of reduced glomerular filtration rate. Cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) have been recently proven to be useful for quantification of CKD. Therefore, we compared the diagnostic value of NGAL with cystatin C and creatinine to evaluate kidney function in hypertensive patients.

Methods:

In this study, 42 hypertensive patients and 30 healthy volunteers were recruited. Serum cystatin C (Scys C) and plasma NGAL were measured using ELISA method. Creatinine, urea, hemoglobin, fibrinogen, and C-reactive protein were measured according to the routine methods. Estimated glomerular filtration rate (eGFR) was considered as the gold standard method (cut-off value of < 78 ml/min/1.73 m².

Results:

In the patient group, plasma NGAL, cystatin C, and creatinine were all significantly correlated with eGFR, and plasma NGAL correlated best with eGFR. Receiver-operating characteristics analysis indicated that plasma NGAL was a better indicator than creatinine and cystatin C for predicting a GFR < 78 ml/min/1.73 m². The sensitivity and specificity for NGAL were 96% and 100%, for cystatin C were 92% and 60% and for creatinine were 76% and 47%, respectively.

Conclusion:

Plasma NGAL demonstrated a higher diagnostic value to detect kidney impairment in the early stages of CKD as compared to Scys C and Scr in hypertensive patients.Key Words: Neutrophil gelatinase-associated lipocalin (NGAL), Cystatin C, Creatinine, Hypertension     

Protective Effects of Interleukin-4 on Tissue Destruction and Morphological Changes of Bovine Nasal Chondrocytes in vitro

Background: Previous studies have shown that some cytokines have protective effects on cartilage in joint diseases. In the current study, effects of IL-4 against morphological changes and tissue degradation induced by IL-1α on bovine nasal cartilage (BNC) explants were investigated. Methods: Fresh BNC samples were prepared from a slaughterhouse under sterile conditions. BNC explants culture was treated with both IL-lα (10 ng/ml) and IL-4 (50 ng/ml) at the same time for 28 days. The morphological characteristics of explants were assessed by using histology techniques and invert microscopy. Matrix metalloproteinase-1 (MMP-1) production was assessed within different days by using Western blotting. Results: IL-lα induced prominent cartilage morphology degradation. The pro and active form of MMP-1 band substantially increased at day 21 of culture. In the presence of both IL-lα and IL-4, chondrocytes preserved their ordinary normal phenotype with intact extracellular matrix. In addition, a significant reduction in pro-MMP-1and inhibition of active MMP-1 was seen. Conclusion: In conclusion, IL-4 could be regarded as a potential candidate in cartilage protecting against the degradation changes of IL-lα. It seems that the preservation effect of IL-4 is associated with significant reduction of MMP-1. Key Words: Chondrocyte, Interleukin-1α, Interleukin-4, Matrix metalloproteinase-1, Bovine nasal cartilage     

Conductive 3D structure nanofibrous scaffolds for spinal cord regeneration

The complex nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials and scaffolds which are capable of stimulating neural tissue repair strategies. Recently, conductive polymers have gained much attention for improving the nerve regeneration. In our previous study, a three-dimensional (3D) structure with reliable performance was achieved for electrospun scaffolds. The main purpose in the current study is formation of electrical excitable 3D scaffolds by appending polyaniline (PANI) to biocompatible polymers. In this paper, an attempt was made to develop conductive nanofibrous scaffolds, which can simultaneously present both electrical and topographical cues to cells. By using a proper 3D structure, two kinds of conductive scaffolds are compared with a non-conductive scaffold. The 3D nanofibrous core-sheath scaffolds, which are conductive, were prepared with nanorough sheath and aligned core. Two different sheath polymers, including poly(lactic-co-glycolic acid) PLGA and PLGA/PANI, with identical PCL/PANI cores were fabricated. Nanofibers of PCL and PLGA blends with PANI have fiber diameters of 234±60.8 nm and 770±166.6 nm, and conductivity of 3.17×10^-5 S/cm and 4.29×10^-5 S/cm, respectively. The cell proliferation evaluation of nerve cells on these two conductive scaffolds and previous non-conductive scaffolds (PLGA) indicate that the first conductive scaffold (PCL/ PANI-PLGA) could be more effective for nerve tissue regeneration. Locomotor scores of grafted animals by developed scaffolds showed significant performance of non-conductive 3D scaffolds. Moreover, the animal studies indicated the ability of two new types of conductive scaffolds as spinal cord regeneration candidates.     

Comparison of the pathogensis of two isolates of <Emphasis Type="Italic">Besnoitia caprae</Emphasis> in inbred BALB/c mice

This study was performed to evaluate the infectivity of bradyzoites of two Besnoitia caprae isolates, BC-1 and BC-2, to inbred BALB/c mice. Each group of inbred BALB/c mice was inoculated intraperitoneally with 1 × 10³, 1 × 10⁴, 1 × 10⁵, 5 × 10⁵ and 1 × 10⁶ of one of the two isolates of B. caprae bradyzoites. The mice were monitored daily for a period of 40 days for survival. After death of each mice, several passages from its peritoneal washing and tissues were analyzed using ribosomal DNA-specific PCR assay. Marked differences in pathogenicity between the isolates were seen. All the inbred BALB/c mice infected with BC-2 survived but all the mice that were administered with 1 × 0⁵, 5 × 10⁵ and 1 × 10⁶ BC-1 bradyzoites were died within 4–9 days post-infection (DPI). Histopathological examination of the tissues of the dead mice revealed hyperemia and necrosis with presence of mononuclear and polymorphonuclear cell infiltration in myocardium, spleen and intestines together with interstitial pneumonia and peritonitis. All inbred BALB/c mice in the 1 × 10³ and 1 × 10⁴ groups of BC-1 inoculated mice survived and they were euthanized after 40 DPI. Chronic inflammation with infiltration of mononuclear cells was evident in myocardium, spleen, alveolar septa of the lungs of most of the examined tissues with hemorrhagic enteritis in the mice infected with 1x10⁶ bradyzoites. The mice infected with different doses of BC-2 were euthanized after 40 DPI and no lesion was seen in histopathological sections of their organs. All peritoneal washings and examined tissues were PCR positive in BC-1 group. This experiment is the first report to show inbred BALB/c mice as a relevant model for B. caprae and demonstrates that this strain of inbred BALB/c mice is a suitable animal model for biological studies and examination of pathogenesis for this species of Besnoitia. The present findings also provide evidence for significant differences between the two isolates of B. caprae.     

DNA vaccine encoding avian influenza virus H5 and Esat-6 of Mycobacterium tuberculosis improved antibody responses against AIV in chickens

The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p < 0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.     

Soil application of entomopathogenic nematodes suppresses the root-knot nematode Meloidogyne javanica in cucumber

Journal of Plant Diseases and Protection - Cucumber (Cucumis sativus) cultivation in commercial greenhouses occupies an important section of vegetable production in Iran. Root-knot nematode...