Inhibitory effects of taraxasterol and aqueous extract of Taraxacum officinale on calcium oxalate crystallization: in vitro study.

OBJECTIVE: We investigated and compared the effects of taraxasterol, aqueous extract of T. officinale (AET) aerial part, and potassium citrate (PC) on calcium oxalate (CaOx) crystallization in vitro. MATERIALS AND METHODS: CaOx crystallization was induced by adding sodium oxalate to synthetic urine. Taraxasterol (2.5, 5, 7.5 and 12.5 µg/mL), extract (1, 2, 4 and 8 mg/mL), and PC (100, 150, 200 and 350 mg/mL) were subjected to anti-crystallization activities. The absorbance and %inhibition of nucleation of CaOx crystals were evaluated by spectrophotometer at 2, 4, 6, 8, 10, 20, 30, 40, 50 and 60 min and the number and morphology of crystals were studied by light microscopy after 60 min. RESULTS: Presence of taraxasterol, extract and PC decreased absorbance in experimental samples compared to control, significantly. The nucleation of crystals is inhibited by taraxasterol, extract, and PC (26-64, 55-63 and 60-70%, respectively). The number of CaOx crystals were decreased in presence of taraxasterol (p < .01), extract (p < .001), and PC (p < .001) in a dose-dependent manner. Presence of taraxasterol, extract, and PC decreased the number of CaC2O4 monohydrate, while increased CaC2O4 dihydrate crystals, significantly. Also, the diameter of CaC2O4 dihydrate crystals was decreased in presence of taraxasterol, extract and PC, significantly. CONCLUSIONS: This research indicated that taraxasterol and extract have anti-crystallization activities and effectiveness of the extract is more potent than taraxasterol. It could be because of another constituent in the extract with the synergistic effect.

Curcumin confers protection to irradiated THP-1 cells while its nanoformulation sensitizes these cells via apoptosis induction.

Protection against ionizing radiation (IR) and sensitization of cancer cells to IR are apparently contrasting phenomena. However, curcumin takes on these contrasting roles leading to either protection or enhanced apoptosis in different irradiated cells. Here we studied whether pretreatment with free curcumin or a novel dendrosomal nanoformulation of curcumin (DNC) could exert protective/sensitizing effects on irradiated THP-1 leukemia cells. We employed assays including MTT viability, clonogenic survival, DNA fragmentation, PI/Annexin V flow cytometry, antioxidant system (ROS, TBARS for lipid peroxidation, 8-OHdG and γH2AX for DNA damage, glutathione, CAT and GPx activity, enzymes gene expression), ELISA (NF-κB and Nrf2 binding, TNF-α release), caspase assay, siRNA silencing of caspase-3, and western blotting to illustrate the observed protective role of curcumin in comparison with the opposite sensitizing role of its nanoformulation at a similar 10 µM concentration. The in vivo relevance of this concentration was determined via intraperitoneal administration in mice. Curcumin significantly enhanced the antioxidant defense, while DNC induced apoptosis and reduced viability as well as survival of irradiated THP-1 cells. Nrf2 binding showed an early rise and fall in DNC-treated cells, despite a gradual increase in curcumin-treated cells. We also demonstrated that DNC induced apoptosis in THP-1 cells via caspase-3 activation; whereas in combination with radiation, DNC alternatively employed a caspase-independent apoptosis pathway involving cytochrome c release from mitochondria.

Nanoformulation of curcumin protects HUVEC endothelial cells against ionizing radiation and suppresses their adhesion to monocytes: potential in prevention of radiation-induced atherosclerosis.

OBJECTIVES: To investigated the potential of a novel dendrosomal nanoformulation of curcumin (DNC) in blocking radiation-induced changes in irradiated human umbilical vein endothelial cells (HUVECs), and their adhesion to human THP-1 monocytoid cells. RESULTS: Co60 gamma rays reduced viability, raised the expression of adhesion molecules, ICAM-1, VCAM-1 and E-selectin (mRNA and protein), augmented the adhesion of THP-1 cells to HUVECs, activated NF-κB binding, increased the release of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) and induced oxidative damage (reduced glutathione declined, while 8-OHdG and TBARS increased). 5 µM DNC significantly inhibited these radiation-induced changes, activated the Nrf-2 pathway, and effectively suppressed THP-1 adhesion to HUVECs, implicating p38 MAPK signaling. CONCLUSION: DNC treatment is a potential preventive method against inflammation and vascular damage from ionizing radiation.

RNA-binding protein Rbm47 binds to Nanog in mouse embryonic stem cells.

Embryonic stem cells (ES cells) are pluripotent cells capable for self-renewal and to differentiate to all cell types. Finding the molecular mechanisms responsible for these unique characteristics of ES cells is important. RNA-binding proteins play important roles in post-transcriptional gene regulation by binding to specific mRNA targets. In this study, we investigated the targets of RNA-binding protein Rbm47 in mouse ES cells. Overexpression of HA epitope-tagged Rbm47 in mouse ES cells followed by RNA-binding protein immunoprecipitation, and then RT-PCR analysis of co-immunoprecipitated RNA showed that Rbm47 binds to Nanog transcript in mouse ES cells and doesn't bind to Sox2 and Oct4 transcripts in these cells. This finding can give rise to reveal molecular mechanisms underlying pluripotency and stemness of ES cells and will be necessary for efficient application of these cells in regenerative medicine and tissue engineering.

MicroRNA signature associated with osteogenic lineage commitment.

Cell-based approaches offer a potential therapeutic strategy for appropriate bone manufacturing. Capable of differentiating into multiple cell types especially osteoblasts spontaneously, unrestricted somatic stem cell (USSC) seems to be a suitable candidate. Recent studies have shown the involvement of microRNAs in several biological processes. miRNA microarray profiling was applied in order to identify the osteo-specific miRNA signature. Prior to this analysis, osteogenic commitment of osteoblasts was evaluated by measuring ALPase activity, biomineralization, specific staining and evaluation of some main osteogenic marker genes. To support our findings, various in silico explorations (for both putative targets and signaling pathways) and empirical analyses (miRNA transfections followed by qPCR of osteogenic indicators and ALPase activity measurement) were carried out. The function of GSK-3b inhibitor was also studied to investigate the role of WNT in osteogenesis. Transient modulation of multiple osteo-miRs (such as mir-199b, 1274a, 30b) with common targets (such as BMPR, TCFs, SMADs) as mediators of osteogenic pathways including cell-cell interactions, WNT and TGF-beta pathways, suggests a mechanism for rapid induction of the osteogenesis as an anti-miRNA therapy. The results of this research have identified the miRNA signature which regulates the osteogenesis mechanism in USSC. To conclude, our study reveals more details about the allocation of USSCs into osteogenic lineage through modulatory effect of miRNAs on targets and pathways required for creating a tissue-specific phenotype and may aid in future clinical interventions.

miRNAs expressed differently in cancer stem cells and cancer cells of human gastric cancer cell line MKN-45.

Recent studies show that cancers may originate from special cells named cancer stem cells (CSCs). As miRNAs have a prominent role in regulating cell activities, a question arise, that is, if there is any difference in miRNA expression level between CSC and other cancer cells of human gastric cancer cell line MKN-45. In this study, CSCs were isolated by fluorescence-activated cell sorter based on the expression level of cell surface marker CD44. CSC characteristics were checked using spheroid formation assay and soft agar assay. Using reverse transcriptase polymerase chain reaction (RT-PCR), the expression level of some stemness genes was studied. Real-time q-PCR was used for analysis of the expression level of miRNAs. CSCs were able to make spheroids and colonies, whereas other cancer cells failed to show aforementioned features. In addition, RT-PCR resulted in a difference in the expression levels of Nanog, Sox2, Lin28 and Oct-4 between these two kinds of cells. Real-time RT-PCR analysis demonstrated an increase in mir-21 and mir-302 expression level in CSCs, relative to cancer cells, whereas let-7a expression level was decreased in CSC in comparison with cancer cells, which may be due to their different differentiation level. On the other hand, mir-372, mir-373 and mir-520c-5p were markedly increased in cancer cells in comparison with CSCs. This study shows that there is a difference in miRNA expression level between CSCs and other cancer cells, which reflects dissimilar molecular pathways in these cells. These miRNAs may be promising objects for targeting CSCs specifically and efficiently.

Down-regulation of miRNA-221 triggers osteogenic differentiation in human stem cells.

Despite interesting in silico evidence, the specific role of mir-221 in osteogenesis has not been studied. We evaluated the osteogenic induction of transient-transfected anti-mir-221 in human unrestricted somatic stem cells and human mesenchymal stem cells both transcriptionally and translationally. In transfected unrestricted somatic stem cells, transcriptions of some osteogenic markers were twice that of the control and translations of osteopontin and osteocalcin were increased from 9 to 39 % and from 0 to 21 %, respectively. Up-regulation of transcribed osteogenic markers in transfected mesenchymal stem cells were 50 times greater than controls while no significant change in translations were observed. Prior to these analyses, the authenticity of stem cells, their osteogenic differentiation and transfection efficiency were verified. Transient modulation of mir-221 therefore suggests a mechanism for rapid induction of osteogenesis as a useful strategy for cell-based therapy.

PRKN, DJ-1, and PINK1 screening identifies novel splice site mutation in PRKN and two novel DJ-1 mutations.

We present results of mutation screening of PRKN gene in 93 Iranian Parkinson's disease (PD) patients with average age at onset (AAO) of 42.2 years. The gene was screened by direct sequencing and by a semi-quantitative PCR protocol for detection of sequence rearrangements. Heterozygous rearrangements were tested by reverse transcription-polymerase chain reaction (RT-PCR). Nine different PRKN mutations were found. One of these, IVS9+1G>A, affects splicing and is novel. Two mutated PRKN alleles were observed in each of 6 patients whose average AAO was 25.7 years. Only 1 patient carried a single mutated allele and his AAO was 41 years. Among patients with AAO of <30 years, 31.3% had two mutated alleles, while only 2.6% with AAO of >30 years carried a PRKN mutation. Analysis of PRKN by RT-PCR led to identification of a novel exon expressed in leukocytes of control and PD individuals. The alternatively spliced transcript if translated would code a protein without a RING Finger 2 domain. Its functional relevance remains to be shown. DJ-I and PINK1 were also screened. Two novel DJ-1 mutations, c.91-2A>G affecting splicing and c.319G>C causing Ala107Pro, were observed among patients with AAO of <31 years, suggesting that PD in a high fraction (>12%) of this group of Iranian patients may be due to mutations in DJ-1. Mutations in PINK1 were not observed. Our results complement previous findings on LRRK2 mutations among Iranian PD patients.

Effective combination of aligned nanocomposite nanofibers and human unrestricted somatic stem cells for bone tissue engineering.

AIM: Bioartificial bone tissue engineering is an increasingly popular technique to solve bone defect challenges. This study aimed to investigate the interactions between matrix composition and appropriate cell type, focusing on hydroxyapatite (HA), to achieve a more effective combination for bone regeneration. METHODS: Human unrestricted somatic stem cells (USSCs) were isolated from placental cord blood. The cellular and molecular events during the osteo-induction of USSCs were evaluated for 21 d under the following conditions: (1) in basal culture, (2) supplemented with hydroxyapatite nanoparticle (nHA) suspension, and (3) seeded on electrospun aligned nanofibrous poly-ɛ-caprolactone/poly-L-lactic acid/nHA (PCL/PLLA/nHA) scaffolds. The scaffolds were characterized using scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR) and tensile test. RESULTS: Maintenance of USSCs for 21 d in basal or osteogenic culture resulted in significant increase in osteoblast differentiation. With nHA suspension, even soluble osteo-inductive additives were ineffective, probably due to induced apoptosis of the cells. In contrast to the hindrance of proliferation by nHA suspension, the scaffolds improved cell growth. The scaffolds mimic the nanostructure of natural bone matrix with the combination of PLLA/PCL (organic phase) and HA (inorganic phase) offering a favorable surface topography, which was demonstrated to possess suitable properties for supporting USSCs. Quantitative measurement of osteogenic markers, enzymatic activity and mineralization indicated that the scaffolds did not disturb, but enhanced the osteogenic potential of USSCs. Moreover, the alignment of the fibers led to cell orientation during cell growth. CONCLUSION: The results demonstrated the synergism of PCL/PLLA/nHA nanofibrous scaffolds and USSCs in the augmentation of osteogenic differentiation. Thus, nHA grafted into PCL/PLLA scaffolds can be a suitable choice for bone tissue regeneration.

A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue.

To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.

Enhanced osteogenic differentiation of cord blood-derived unrestricted somatic stem cells on electrospun nanofibers.

A new stem cell-scaffold construct based on poly-L-lactide (PLLA) nanofibers grafted with collagen (PLLA-COL) and cord blood-derived unrestricted somatic stem cells (USSC) were proposed to hold promising characteristics for bone tissue engineering. Fabricated nanofibers were characterized using SEM, ATR-FTIR, tensile and contact angle measurements. The capacity of PLLA, plasma-treated PLLA (PLLA-pl) and PLLA-COL scaffolds to support proliferation and osteogenic differentiation of USSC was evaluated using MTT assay and common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium mineral deposition and bone-related genes. All three scaffolds showed nanofibrous and porous structure with suitable physical characteristics. Higher proliferation and viability of USSC was observed on PLLA-COL nanofibers compared to control surfaces. In osteogenic medium, ALP activity and calcium deposition exhibited the highest values on PLLA-COL scaffolds on days 7 and 14. These markers were also greater on PLLA and PLLA-pl compared to TCPS. Higher levels of collagen I, osteonectin and bone morphogenetic protein-2 were detected on PLLA-COL compared to PLLA and PLLA-pl. Runx2 and osteocalcin were also expressed continuously on all scaffolds during induction. These observations suggested the enhanced proliferation and osteogenic differentiation of USSC on PLLA-COL nanofiber scaffolds and introduced a new combination of stem cell-scaffold constructs with desired characteristics for application in bone tissue engineering.

Efficient refolding of recombinant lipase from Escherichia coli inclusion bodies by response surface methodology.

An experimental design was employed to optimize the refolding conditions of a recombinant lipase from Pseudomonas sp. which expressed as inclusion bodies in Escherichia coli. The effects of several variables on the refolding and activation of the enzyme has been studied. Because of the complexity of the reaction with respect to the number of parameters that can affect the refolding efficiency, 2(6-1) half-fractional factorial design (H-FFD) was employed for initial screening of the factors, potentially influencing the response. Experiments were performed in triplicate at two levels. Subsequently, the selected factors were subjected to response surface methodology (RSM) with a four factor-five coded level central composite design (CCD), using Quadratic model for obtaining the optimum values for the factors. The adequacy of the calculated model was confirmed by the coefficient of determination (R(2)) and F value of 0.89 and 9.12, respectively. The optimized condition for the refolding was obtained in the refolding buffer containing unfolded lipase (10 microg/ml) and foldase (3 microg/ml) in combination with glycerol (10%), NaCl (1M) and sucrose (0.5M). Using chemicals in combination with foldase under the optimal condition exhibited a 50% increase in refolding yield over the conventional method.

High-level expression of lipase in Escherichia coli and recovery of active recombinant enzyme through in vitro refolding.

Microbial lipases are widely used for biotechnological applications. In this study we have cloned and sequenced the lipase and lipase specific foldase genes of a Pseudomonas sp., which was isolated from Southern Iran. The lipase was composed of 936 bp which encoded 311 amino acids and the lipase specific foldase gene consisted of 1008 bp which encoded 336 amino acids. The low amount of recombinant lipase was expressed as an active enzyme in Escherichia coli harboring a plasmid with the clustered lipase and lipase specific foldase genes. To increase the enzyme expression level, the lipase and lipase specific foldase genes subcloned into two separate expression vectors. The lipase was expressed as inactive inclusion bodies under the control of the strong T7 promoter. Inclusion bodies were dissolved in 8M urea and 1mM dithiothreitol (DTT) and purified using Ni-nitrilotriacetic acid column. Subsequently, purified lipase diluted in 20mM phosphate buffer (pH 7) containing the lipase specific foldase which was expressed in another clone of E. coli. In the presence of foldase, it was possible to achieve active lipase with a specific activity of up to 240 IU/mg using a simple refolding procedure. Moreover, the effect of different concentrations of various additives was investigated on the refolding of denatured lipase. The best yield of 70 IU/ml with the specific activity of 3000 IU/mg were obtained after incubation of denatured enzyme in a refolding buffer containing lipase specific foldase (0.005 mg/ml), 1M NaCl and 10% glycerol at 4 degrees C.

Nanohydroxyapatite-coated electrospun poly(l-lactide) nanofibers enhance osteogenic differentiation of stem cells and induce ectopic bone formation.

A combination of calcium phosphates with nanofibrous scaffolds holds promising potential for bone tissue engineering applications. In this study, nanohydroxyapatite (n-HA) was coated on the plasma-treated surface of electrospun poly(l-lactide) (PLLA) nanofibers and the capacity of fabricated scaffolds for bone formation was investigated in vitro using human cord blood derived unrestricted somatic stem cells (USSC) under osteogenic induction and in vivo after subcutaneous implantation. PLLA and n-HA-coated PLLA (n-HA/PLLA) scaffolds exhibited a nanofibrous structure with interconnected pores and suitable mechanical properties. These scaffolds were also shown to support attachment, spreading, and proliferation of USSC, as shown by their flattened normal morphology and MTT assay. During osteogenic differentiation, significantly higher values of ALP activity, biomineralization, and bone-related gene expression were observed on n-HA/PLLA compared to PLLA scaffolds. Subsequently, these markers were measured in higher amounts in USSC on PLLA nanofibers compared to TCPS. According to the in vivo results, ossification and formation of trabeculi was observed in the n-HA/PLLA scaffold compared to PLLA. Taking together, it was shown that nanofibrous structure enhanced osteogenic differentiation of USSC. Furthermore, surface-coated n-HA stimulated the effect of nanofibers on the orientation of USSC toward osteolineage. In addition, the n-HA/PLLA electrospun scaffold showed the capacity for ectopic bone formation in the absence of exogenous cells.

Binding of Tris to Bacillus licheniformis alpha-amylase can affect its starch hydrolysis activity.

Bacillus licheniformis alpha-amylase (BLA) is routinely used as a model thermostable amylase in biochemical studies. Its starch hydrolysis activity has recently been studied in Tris buffer. Here, we address the question that whether the application of Tris buffer may influence the results of BLA activity analyses. Based on the inhibition studies and docking simulations, we suggest that Tris molecule is a competitive inhibitor of starch-hydrolyzing activity of BLA, and it has a high tendency to bind the enzyme active site. Hence, it is critically important to consider such effect when interpreting the results of activity studies of this enzyme in Tris buffer.

Green synthesis of manganese nanoparticles: Applications and future perspective-A review.

Nanobiotechnology is a promising and appearing field of nanotechnology. In recent years, the necessity of making biocompatible materials for different applications in various area such as health, medicine, water treatment and purification, etc. caused more attention to this area. Today, green synthesis of different nanoparticles (NPs) has been extensively studied. However, less attention has been paid to manganese as a high-performance metal in various applications such as medicine, biomedicine, biosensors, water treatment and purification, electronics, electrochemistry, photoelectronics, catalysis, and etc. Manganese oxides (Mn-oxides) has wealthy structures such as MnO, Mn5O8, Mn2O3, MnO2, and Mn3O4, and can be used in a variety of fields. Mn-oxide NPs potentially hold great promise for sustainable nanotechnology. This review focusses on the green synthesis, applications and future perspective of Mn NPs. Different methods of green synthesis of Mn NPs, including synthesis using plant extract, synthesis using microorganism, and low-temperature synthesis of Mn NPs have been investigated and presented. Structure, and size, of green synthesized Mn NPs via each method have been compared. Also, various applications of the green synthesized Mn NPs have been reviewed. Furthermore, the future perspective of green synthesis and applications of the green synthesized Mn NPs are expressed. Also, different applications explained for green synthesized Mn NPs are expressed as well as potential applications for green synthesized Mn NPs are suggested.

Surface modification of PES membrane via aminolysis and immobilization of carboxymethylcellulose and sulphated carboxymethylcellulose for hemodialysis.

Sulphated carboxymethylcellulose (SCMC) was synthesized and characterized via FT-IR and CHNS analysis. The PES membranes were prepared and modified via surface amination with an aminolysis reaction and amount of amino groups was measured. The carboxymethylcellulose (CMC) and SCMC were immobilized on the surface of the aminated PES membranes (PES-NH2) via amide bonds to synthesize PES-CMC and PES-SCMC membranes, respectively, and the concentration of immobilized CMC and SCMC was determined. The unmodified PES, PES-CMC, and PES-SCMC membranes were characterized in terms of morphology, overall porosity, mean pore size, zeta potential, tensile strength, contact angle, protein adsorption and platelet adhesion. The results showed a decrease in contact angle, protein adsorption and platelet adhesion in the case of PES-CMC and PES-SCMC compared to unmodified PES membranes, which supported the increased hemocompatibility of the modified membranes especially for the PES-SCMC membrane. Moreover, the PES-CMC and PES-SCMC membranes displayed good antifouling properties, especially for PES-SCMC.

Optimisation of green synthesis of MnO nanoparticles via utilising response surface methodology.

This study concerns the optimisation of green synthesis of manganese oxide nanoparticles (MnO NPs) with Dittrichia graveolens (L.) extract via response surface methodology (RSM). Central composite design was used to evaluate the effect of pH, time, and the extract to the metal ratio on the synthesised nanoparticles (NPs). Nine runs were designed to investigate the effect of each parameter while NPs were synthesised under different conditions. Considering the p-values (p-value < 0.05), it is indicated that the extract to the metal ratio was the most effective parameter. The synthesised NPs were characterised using UV-vis. Synthesis of the NPs by polyphenolic compounds of green reducing agent and their stabilisation by curcumin was confirmed by Fourier transform infrared spectra and the surface morphology of the spherical MnO NPs was studied by field-emission scanning electron microscopy and transmission electron microscope techniques. The present researchers claimed the optimal condition as follows: time = 56.7 min, pH = 7.2, and the extract to the metal ratio = 87.9 v/v. MnO NPs at optimum condition were then employed for degradation of industrial dyes and they showed high dye degradation activity against Rhodamine B and light green dye. The average size of the synthesised MnO NPs at optimal condition was claimed to be nearly 38 nm.

Antiurolithiatic effect of the taraxasterol on ethylene glycol induced kidney calculi in male rats.

Taraxasterol is one of the important constituents of Taraxacum officinale L. (Compositae) with antioxidant potential. The present study was designed to evaluate and compare the antiurolithiatic effects of taraxasterol and potassium citrate in the ethylene glycol induced urolithiatic rat. Urolithiasis was induced by ammonium chloride and ethylene glycol in adult male rats. Taraxasterol (2, 4 and 8 mg/kg) and potassium citrate (2.5 g/kg) were treated for 33 days by gavage. Then, the animals were anesthetized and weighted and blood, urine, liver and kidney sampling were done. The kidney sections were prepared by hematoxylin & eosin staining. The liver and kidney coefficients, urine pH, calcium, magnesium, oxalate and citrate levels, serum albumin, calcium and magnesium levels, serum alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities, superoxide dismutase and glutathione peroxidase activities in serum, kidney and liver, number of calcium oxalate crystal deposits, score of crystal deposits, score of histopathological damages and score of inflammation in kidney sections were evaluated. The results showed that taraxasterol decreased liver and kidney coefficients (p < 0.001), serum calcium (p < 0.01) level, serum alanine aminotransferase (p < 0.001), aspartate aminotransferase (p < 0.001), lactate dehydrogenase (p < 0.05) activities, urine magnesium (p < 0.05) and oxalate (p < 0.001) levels, number of crystal deposits (p < 0.001), score of crystal deposits (p < 0.01), score of histopathological damages (p < 0.001) and score of inflammation (p < 0.01) in kidney sections, while increased urine pH (p < 0.01), calcium (p < 0.001) and citrate (p < 0.05), serum magnesium (p < 0.001) and albumin (p < 0.01) levels, superoxide dismutase and glutathione peroxidase in serum (p < 0.01), kidney (p < 0.05 and p < 0.001, respectively) and liver (p < 0.01 and p < 0.001, respectively) tissue homogenates in treated urolithiatic rats in comparison to the control urolithiatic rats. The effect of potassium citrate is the same as taraxasterol in treated urolithiatic rats. In conclusion, the effect of taraxasterol could be by improving liver function, changing serum and urine parameters, maintaining the antioxidant environment, reducing crystal deposition, excretion of small deposits from kidney and reducing the chance of them being retained in the urinary tract.

Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization.

In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.