l-asparaginase production and enhancement by Sarocladium strictum: In vitro evaluation of anti-cancerous properties.

AIMS: Utilization of l-asparaginase has been one of the effective strategies for the treatment of lymphoblastic leukaemia. Since the currently used bacterial l-asparaginase causes side effects, searching for new enzyme sources has been an active field of research. This study focuses on the characterization of an l-asparaginase-producing fungal strain. METHODS AND RESULTS: Sarocladium strictum was identified as a potent enzyme-producing strain. For the enhancement of enzyme production, we used two-level factorial design and response surface methodology. The optimization of significant factors showed a 1·84-fold increase in enzyme production. The Km and Vmax values of the enzyme were 9·74 mmol l-1 and 8·19 µmol min-1 . The toxicity of the produced l-asparaginase was measured on K562 and HL60 cancer cell lines and L6 as normal cells. The IC50 values were calculated as 0·4 and 0·5 IU ml-1 for K562 and HL60 respectively and no significant effect was observed in L6. BrdU proliferation and caspase-3 activity assay in l-asparaginase treated HL60 and K562 cells indicated that cell proliferation rates and apoptotic cell death were reduced. CONCLUSIONS: The cytotoxic properties of the produced fungal enzyme indicated significant growth inhibition in cancer cells while having a little toxic effect on normal cells. The possibility of mass production alongside having suitable cytotoxic and kinetic properties suggest the probable use of the produced l-asparaginase for further researches as a potential chemotherapeutic agent. SIGNIFICANCE AND IMPACT OF THE STUDY: The lack of significant l-glutaminase activity and promising toxicity properties in S. strictum and the closer evolutionary relativeness of fungi enzymes to human enzymes compared to bacterial enzymes suggest a new source with lower toxicity and anti-cancerous properties, causing less side effect problems.

Effects of different sources of nitrogen on performance, relative population of rumen microorganisms, ruminal fermentation and blood parameters in male feedlotting lambs.

Slow-release urea (SRU) can substitute dietary protein sources in the diet of feedlotting ruminant species . However, different SRU structures show varying results of productive performance. This study was conducted to investigate the effect of different sources of nitrogen on performance, blood parameter, ruminal fermentation and relative population of rumen microorganisms in male Mehraban lambs. Thirty-five male lambs with an average initial BW of 34.7 ± 1.8 kg were assigned randomly to five treatments. Diets consisted of concentrate mixture and mineral and vitamin supplements plus (1) alfalfa and soybean meal, (2) wheat straw and soybean meal, (3) wheat straw and urea, (4) wheat straw and Optigen® (a commercial SRU supplement) and (5) wheat straw and SRU produced in the laboratory. No statistical difference was observed in animal performance and DM intake among treatments. The mean value of ruminal pH and ammonia was higher (P < 0.05) for the SRU diet compared with WU diet. The difference in pH is likely to be due to the higher ammonia level as VFAs concentrations were unchanged. The level of blood urea nitrogen (BUN) was different among treatments (P = 0.065). The highest concentration of BUN was recorded in Optigen diet (183.1 mg/l), whereas the lowest value was recorded in wheat straw-soybean meal diet (147 mg/l). The amount of albumin and total protein was not affected by the treatments. The relative population of total protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus in the SRU treatment was higher (P < 0.01) than that in urea treatment at 3 h post-feeding. During the period of lack of high-quality forage and in order to reduce dietary costs, low-quality forage with urea sources can be used in the diet. Results of microbial populations revealed that SRU can be used as a nitrogen source which can sustainably provide nitrogen for rumen microorganism without negative effects on the performance of feedlotting lambs.

Characterization of pyrene degradation and metabolite identification by Basidioascus persicus and mineralization enhancement with bacterial-yeast co-culture.

In this paper, we introduce pyrene degrader halotolerant yeast, Basidioascus persicus EBL-C16 and characterize its growth in different salt concentrations. Investigation of the removal of different concentrations of pyrene showed that B. persicus EBL-C16 was able to eliminate 50-90% of pyrene in the presence of 2.5% salt and also mineralize phenanthrene and anthracene. Growth and depletion kinetics of 500 mg L-1 of pyrene removal revealed 79% degradation and the growth rate reached 1.4 g L-1 of dry weight, decomposition constant rate was obtained as 0.074 day-1 and the half-life was 9.33 days. When minimal medium was replaced with Persian Gulf water, 48% increase in pyrene removal was detected by yeast strain. The mass spectrometry of the treated samples specified the phthalic acid pathway as the metabolic pathway of pyrene degradation by B. persicus. Study of the synergistic effect of using rhamnolipid and co-culture of yeast with Pseudomonas putida ATCC 12633 revealed that the combination of both of them with B. persicus increased 21% pyrene elimination. The findings of this study can be used to comprehend the mechanisms of oil hydrocarbon degradation by yeasts. Furthermore, the results demonstrated the promising potential of yeast-bacteria co-culture for cleaning of oil spills in marine and saline soil contaminated areas.

Investigating the effects of size, charge, viscosity and bilayer flexibility on liposomal delivery under convective flow.

Convective flow is one of the main mechanisms of mass transfer employed in drug delivery (e.g. osmotic pumps) and working in material transport in the body (e.g. blood circulation). Although convective flow has been investigated extensively, not much data is available on convective behavior of nanoparticles, the subject of the present investigation. Here, liposomes with different sizes, charges, bilayer flexibilities and medium viscosities were encountered convective flow and changes in their properties were monitored over 48h. For large particles (>350nm), neutral liposomes (NL) showed significant phase separation and decreased lipid content over time, while positively or negatively charged liposomes remained homogenous. Reduction of size in NLs to about 100nm resolved phase separation problem, but their lipid content still showed reduction; no such a problem was observed in charged small liposomes. When bilayer flexibility of large NLs was increased, neither phase separation nor decreased lipid content was observed. Increasing medium viscosity for large NLs from 3.4cP to 45.2cP again solved the problem and a uniform liposomal delivery was observed. These results indicate that size, charge, bilayer flexibility and viscosity affect convective liposomal delivery and that uniform delivery is possible even in large liposomes by optimizing such factors.

Enhancement and in vitro evaluation of amifostine permeation through artificial membrane (PAMPA) via ion pairing approach and mechanistic selection of its optimal counter ion.

This study presents the results of in vitro evaluation of a series of organic counter ions that form ion pairs with amifostine. The selected counter ions have different lipophilicity, shape and flexibility. Intrinsic octanol buffer partition coefficient and binding constant of the ion pairs were calculated using quasi-equilibrium analysis. Permeation through hydrophobic PAMPA membranes of amifostine and its ion pairs with different counter ions was studied. Three counter ions, succinic acid, benzoic acid and phthalic acid demonstrated an increase in the apparent partition coefficient of amifostine in n-octanol. These counter ions were selected for permeability experiments in PAMPA membranes and an increase of the apparent permeability value Papp (cm/s) was also observed as a function of the counter ion concentration. Phthalic acid produced 1.6-fold increase of log PAB while for benzoic acid and succinic acid the values were 1.2 and 0.75-fold respectively. PAMPA permeability of amifostine significantly increased in the presence of phthalic acid (42-fold), benzoic acid (37-fold) and succinic acid (10.5-fold). This study showed that the permeability of amifostine across a lipophilic membrane was enhanced in the presence of counter-ions resulting ion pair formation.

The influence of lipid composition and surface charge on biodistribution of intact liposomes releasing from hydrogel-embedded vesicles.

Mixed drug delivery systems possess advantages over discrete systems, and can be used as a strategy to design more effective formulations. They are more valuable if the embedded particles perform well, rather than using drugs that have been affected by the surrounding vehicle. In order to address this concept, different liposomes have been incorporated into hydrogel to evaluate the potential effect on the controlled release of liposomes. Radiolabeled liposomes, with respect to different acyl chain lengths (DMPC, DPPC, or DSPC) and charges (neutral, negative [DSPG], or positive [DOTAP]) were integrated into chitosan-glycerophosphate. The results obtained from the biodistribution showed that the DSPC liposomes had the highest area under the curve (AUC) values, both in the blood (206.5%ID/gh(-1)) and peritoneum (622.3%ID/gh(-1)), when compared to the DPPC and DMPC formulations, whether in liposomal hydrogel or dispersion. Interesting results were observed in that the hydrogel could reverse the peritoneal retention of negatively charged liposomes, increasing to 8 times its AUC value, to attain the highest amount among all formulations. The interactions between the liposomes and chitosan-glycerophosphate, confirmed by the Fourier transform infrared (FTIR) spectra as shifted characteristic peaks, were observed in the combined systems. Overall, the hydrogel could control the release of intact liposomes, which could be manipulated by both the liposome type and interactions between the two vehicles.

Development of a novel lipidic nanoparticle probe using liposomal encapsulated Gd2O3-DEG for molecular MRI.

Recently, it has been showed that gadolinium oxide nanoparticles can provide high-contrast enhancement in magnetic resonance imaging (MRI). Moreover, liposomes due to high biocompatibility have shown unique model systems, with the most successful application being the drug delivery system. As a suitable cell-tracking contrast agent (CA) in molecular MRI (mMRI), the synthesis and optimisation characteristic of a novel paramagnetic liposomes (PMLs) based on gadolinium nanoparticles, essentially composed of a new complex of gadolinium oxide-diethylene glycol (Gd2O3-DEG) loaded in liposomes have been determined in this research. Gd2O3-DEG was prepared by a new supervised polyol method and was encapsulated with liposome by the film hydration method. The paramagnetic liposome nanoparticle (PMLN) sizes ranged from 65 to 170 nm. The r1 of PMLNs and Gd2O3-DEG were much higher than that of Gd-diethylenetriamine penta-acetic acid (Gd-DTPA). In MC/9 cell lines, the experiments showed similar results as in water. PMLNs with lower T1 than Gd-DTPA are sensitive, positive MRI CA that could be attractive candidates for cellular and molecular lipid content targets such as diagnostic applications.

Hydrogel-embeded vesicles, as a novel approach for prolonged release and delivery of liposome, in vitro and in vivo.

A novel delivery concept based on the integration of liposomes in hydrogel for the controlled release of liposomes was developed. As an in situ forming hydrogel, chitosan-glycerophosphate was used and gelation time at different temperatures was studied. Liposomes (DSPC/chol/DOPE) were labelled with (99m)Tc-hexamethylpropyleneamineoxime ((99m)Tc-HMPAO). (99m)Tc-HMPAO solution, hydrogel/(99m)Tc-HMPAO, (99m)Tc-HMPAO liposomes and hydrogel/(99m)Tc-HMPAO liposomes were injected into mouse peritoneum. The percentages of radioactive injected dose per gram of tissue (%ID/g) and %ID of peritoneum lavage were obtained. Results showed that free label left the peritoneal cavity rapidly in both solution and hydrogel forms, such that the activity was 2.5 and 3.8 (%ID) after one hour, respectively. The values for liposomes and liposomal hydrogel were 25.8 and 51.2 (%ID) and decreased to 1.9 and 19.2 after 24 h, respectively. The blood profile of liposomal hydrogel showed a two-phase profile including a descending trend in early hours regarding gel formation followed by an ascending trend due to gel disappearance by time. Free label had high activity in reticuloendothelial system (RES) and the gastrointestinal tract during the early hours and then dropped. In contrast, the accumulation of liposomes increased in RES during 24 h in the range of 1-34.5 and 1.1-35.1 (%ID/g) for plain liposomes and liposomal hydrogel, respectively. Overall, incorporation of liposomes in hydrogel could be a useful strategy to prolong the release of liposomes.

Sequence Analysis of Different Domains of Plasmodium vivax Apical Membrane Antigen (PvAMA-1 gene) Locus in Iran.

BACKGROUND: Plasmodium vivax is responsible for approximately 80 million malaria cases in the world. Apical membrane antigen1 (AMA-1) is a type I integral membrane protein present in all Plasmodium species. AMA-1 interferes in critical steps of invasion of human hepatocytes by sporozoites and red blood cells by merozoites and is one of the most immunodominant antigens for eliciting a protective immune response in human. It is considered as a promising antigen for inclusion in a vaccine against P. vivax. Since more knowledge is needed to lighten the scope of such antigen we compared genetic variation in P. vivax AMA-1from an Iranian isolate with those reported from some of the other malarious countries so far. METHODS: P. vivax genomic DNA was extracted from the whole blood of an Iranian patient with patent P. vivax infection. The nucleotide sequence for 446 amino acid (AA) residues (42-488 of PvAMA-1) was amplified by PCR and cloned in pUC19 vector for sequencing. RESULTS: Sequence analysis of the antigen showed a high degree of identity (99%) with strong homology to the PvAMA-1 gene of P. vivax S3 and SKO814 isolates from India and Korea (Asian isolates) respectively, and 96% similarity with P. vivax Sal-1 AMA-1 gene from El Salvador. CONCLUSIONS: We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries.

Enhancement by terpenes of 5-fluorouracil permeation through the stratum corneum: model solvent approach.

5-Fluorouracil permeates the stratum corneum through the intercellular pathway. 5-Fluorouracil is hydrophilic and, therefore, its partitioning from the aqueous region into the hydrocarbon interior of stratum corneum lipids is expected to be an important stage of its permeation and a target for some permeation enhancers. It has also been reported that complexation plays a role in the enhancement effect of some accelerants. These mechanisms have been investigated. For partitioning-permeation studies, isooctane was chosen as a model of the hydrocarbon interior of stratum corneum lipid bilayers and the effects of 26 different terpene enhancers on the solubility of 5-fluorouracil in isooctane were measured. Results were then compared with the effects of the same enhancers on the permeation of 5-fluorouracil through the epidermis in man. The stoichiometry of interaction of cineole and limonene with 5-fluorouracil were also studied to reveal possible complex formation. Solubility studies revealed good correlation between solubility and enhancement ratios for the majority of terpenes, indicating that one mechanism by which terpenes increase permeation of the stratum corneum by 5-fluorouracil is by improvement of partitioning. Stoichiometry studies showed that cineole can form 1:1 or higher complexes with 5-fluorouracil. With limonene, only a weak 1:1 complex was indicated. Data obtained using epidermis from man show that the enhancement effect of cineole toward 5-fluorouracil is much higher than that of limonene. These data reveal that terpenes might increase the permeation of 5-fluorouracil through the stratum corneum as a result of complex formation and a form of facilitated transport.