Phagemid Research Articles

Feasibility of Generating Peaks of Bioaerosols for Laboratory Experiments

Bioaerosol concentration peaks are frequently encountered in real-life atmospheres (indoor, outdoor, workplace, etc.), where they can be caused by several factors. However, evolution over time and variability of microbiological pollutant concentrations remain under-documented. The contribution of such peaks in the onset or the worsening of respiratory symptoms – in particular immuno-allergic reactions – has yet to be extensively studied. Although experimental bioaerosol generators are increasingly used, the intentional and controlled production of concentration peaks of biological agents has not yet been utilized in laboratory experiments. The main objective of this study was to show that it is possible to produce experimental bioaerosol concentration peaks with defined characteristics. Experiments were performed with a ‘Liquid Sparging Aerosoliser’-type generator. With this system, peaks can be created by increasing the bubbling airflow through a film of bacterial (Escherichia coli) or fungal (Penicillium brevicompactum) liquid culture. The higher the set point value of the bubbling airflow, the greater the maximum bioaerosol concentration during the generated peak. Similarly, longer-lived peaks can be created by maintaining the increased airflow for a longer period of time. For both studied species, the relative size distribution was constant over time, regardless of modifications to the bubbling flow rate. The operator can monitor and control peak formation with this system thanks to real-time measurement of the number concentrations, targeting the appropriate particle size classes corresponding to diameters of the aerosolised microorganisms. This generator, characterized by gentle aerosolisation and a capacity to produce bioaerosol peaks, may contribute to enrich laboratory experiments for numerous applications.

Two newPenicilliumspeciesPenicillium buchwaldiiandPenicillium spathulatum, producing the anticancer compound asperphenamate

Penicillium buchwaldii sp. nov. (type strain CBS 117181(T) =IBT 6005(T) =IMI 30428(T) ) and Penicillium spathulatum sp. nov. (CBS 117192(T) =IBT 22220(T) ) are described as new species based on a polyphasic taxonomic approach. Isolates of P.buchwaldii typically have terverticillate conidiophores with echinulate thick-walled conidia and produce the extrolites asperphenamate, citreoisocoumarin, communesin A and B, asperentin and 5'-hydroxy-asperentin. Penicillium spathulatum is unique in having restricted colonies on Czapek yeast agar (CYA) with an olive grey reverse, good growth on CYA supplemented with 5% NaCl, terverticillate bi- and ter-ramulate conidiophores and consistently produces the extrolites benzomalvin A and D and asperphenamate. The two new species belong to Penicillium section Brevicompacta and are phylogenetically closely related to Penicillium tularense. With exception of Penicillium fennelliae, asperphenamate is also produced by all other species in section Brevicompacta (P.tularense, Penicillium brevicompactum, Penicillium bialowiezense, Penicillium olsonii, Penicillium astrolabium and Penicillium neocrassum). Both new species have a worldwide distribution. The new species were mainly isolated from indoor environments and food and feedstuffs. The fact that asperphenamate has been found in many widely different plants may indicate that endophytic fungi rather than the plants are the actual producers.

Optimized Production and Characterization of ?-fructofuranosidase from Penicillium brevicompactum

The present study is aimed at the optimization of ?-fructofuranosidase from Penicillium brevicompactum using orange fruit peel waste as a substrate in Czapek Dox as basal medium by submerged fermentation (SmF). The optimum pH, temperature and incubation period for enzyme production were 5, 30°C and 4th day respectively. Of the carbon source, sucrose was recorded to be the best carbon source for enzyme production yeast extract was ideal nitrogen source for the ?-fructofuranosidase production. The enzyme was purified to 5.8 fold with recovery of 3.2% by DEAE-column chromatography and the molecular weight was estimated to be 64 KDa by SDS-PAGE. It has a Vmax value of 15.8 U/mg and Km value of 0.23 mg/ml at pH 6. The enzyme activity was found to be stable at 50°C for 30 minutes and it was stimulated by metal ions like Na+ and Ca2+ and inhibited by Zinc. These results highlight the industrial potentials of the substrates as possible raw materials for ?-fructofuranosidase enzyme production from Penicillium brevicompactum DOI: http://dx.doi.org/10.3329/bjm.v28i1.11803 Bangladesh J Microbiol, Volume 28, Number 1, June 2011, pp 13-18

Microscopic fungi recovered from honey and their toxinogenity

The objective of this investigation was to contribute towards the knowledge of microbiology of honey, more than 50 samples of honey from Slovakia and other countries were mycologically investigated in terms of the overall fungal diversity and toxicological potential of isolated fungi from Penicillium genera. The study revealed that out of 13 genera recovered, Penicillium was the most frequent and diverse genus, followed by Aspergillus and Cladosporium being found in 65.91 % (29 samples), 34.1 % (15 samples) and 29.55 % (13 samples), respectively. The most frequently encountered taxa from Penicillium genera were Penicillium chrysogenum (found in 22.73 %), Penicillium brevicompactum (13.64 %), Penicillium crustosum (11.36 %) and Penicillium griseofulvum (11.36 %). In addition, the following genera were recorded (in descending order) Mycelia (18.18 %), Fusarium (11.36 %), Mucor (9.09 %), Acremonium (6.82 %), Alternaria (4.55 %), Epicoccum (4.55 %), and finally Botrytis, Eurotium Trichoderma and Phoma all were encountered in 2.27 % of the samples being represented. The mean value counts of total fungi ranged from 0.00 to 2 × 102 cfu.g−1. Outcomes from mycotoxin screening within the appropriate potentially toxinogenic species from Penicillium genera showed a number of mycotoxin producers, namely those forming citrinin (n = 1), cyclopiazonic acid (n = 5), griseofulvin (n = 5), patulin (n = 5), penitrem A (n = 2) and roquefortin C (n = 13).

Degradation of terbuthylazine, difenoconazole and pendimethalin pesticides by selected fungi cultures

Contamination of waters by xenobiotic compounds such as pesticides presents a serious environmental problem with substantial levels of pesticides now contaminating European water resources. The aim of this work was to evaluate the ability of the fungi Fusarium oxysporum, Aspergillus oryzae, Lentinula edodes, Penicillium brevicompactum and Lecanicillium saksenae, for the biodegradation of the pesticides terbuthylazine, difenoconazole and pendimethalin in batch liquid cultures. These pesticides are common soil and water contaminants and terbuthylazine is considered the most persistent triazine herbicide in surface environments.P. brevicompactum and L. saksenae were achieved by enrichment, isolation and screening of fungi capable to metabolize the pesticides studied. The isolates were obtained from two pesticide-primed materials (soil and biomixture).Despite the relatively high persistence of terbuthylazine, the results obtained in this work showed that the fungi species studied have a high capability of biotransformation of this xenobiotic, comparatively the results obtained in other similar studies. The highest removal percentage of terbuthylazine from liquid medium was achieved with A. oryzae (~80%), although the major biodegradation has been reached with P. brevicompactum. The higher ability of P. brevicompactum to metabolize terbuthylazine was presumably acquired through chronic exposure to contamination with the herbicide. L. saksenae could remove 99.5% of the available pendimethalin in batch liquid cultures.L. edodes proved to be a fungus with a high potential for biodegradation of pesticides, especially difenoconazole and pendimethalin.Furthermore, the metabolite desethyl-terbuthylazine was detected in L. edodes liquid culture medium, indicating terbuthylazine biodegradation by this fungus.The fungi strains investigated could prove to be valuable as active pesticide-degrading microorganisms, increasing the efficiency of biopurification systems containing wastewaters contaminated with the xenobiotics studied or compounds with similar intrinsic characteristics.

Assessment of lactobacilli strains as yogurt bioprotective cultures

Eleven antifungal Lactobacillus strains previously isolated from cow and goat milk were fully characterized using molecular and phenotypic methods. Their antifungal activities were tested in milk and yogurt, against fungal species (Debaryomyces hansenii, Kluyveromyces lactis, Kluyveromyces marxianus, Penicillium brevicompactum, Rhodotorula mucilaginosa and Yarrowia lipolytica) commonly involved in the spoilage of dairy products. The antifungal strains belonged to Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus zeae and Lactobacillus harbinensis species and showed different acidifying and growth capacities in milk. All tested Lactobacillus strains showed an antifungal activity in milk with strain-dependent activity spectra. Lb. harbinensis showed a very strong antifungal effect in yogurt by completely inhibiting all tested fungi as compared to control. The other tested strains were much less effective. It is the first time that the antifungal activity of Lb. harbinensis is described. This strain is a potential candidate for yogurt biopreservation.

Applications of Ultrasound to Enhance Mycophenolic Acid Production

Reducing production costs for fermentation-based drugs (e.g., antibiotics) is crucial for the long-term sustainability of healthcare. In this study, we propose a novel low-intensity pulsed ultrasound (LIPUS) stimulation scheme using a nominal frequency of 1.5 MHz with a 20% duty cycle (200 μs ultrasound on and 800 μs ultrasound off) to increase production of fermentation-based drugs. We chose Penicillium brevicompactum as a model system to demonstrate the performance of our LIPUS system. Penicillium brevicompactum can produce mycophenolic acid (MPA), an immunosuppressive agent commonly used to prevent rejection after organ transplantation. We have stimulated Penicillium brevicompactum in 50 mL shake flasks using LIPUS during its fermentation. After a series of screening experiments to optimize ultrasound parameters (e.g., ultrasound intensities, treatment duration and treatment frequency per day), it was concluded that ultrasound stimulation can increase MPA production by as much as 60% when treated eight times a day for 10-min durations at an intensity (spatial peak temporal average) of 200 mW/cm2. These findings elucidate a new approach to reduce the cost of producing fermentation-based drugs.

Association between the synovial expression of cyclic citrullinated peptide and susceptibility variants of HLA-DRB1 shared epitope alleles and PADI 4 gene single nucleotide polymorphisms in patients with rheumatoid arthritis

To analyze the association between the synovial expression of cyclic citrullinated peptide (CCP) and susceptibility variants of HLA-DRB1 shared epitope (SE) alleles and/or peptidylarginine deiminase 4 (PADI4) gene single nucleotide polymorphisms (SNP) in patients with rheumatoid arthritis (RA). From October 2008 to December 2011, 53 RA patients and 42 controls were enrolled. The expression of CCP in RA synovial tissues was detected by immunohistochemical assay with 6×His tagged anti-CCP single chain fragment V (ScFv) antibodies generated by pHEN2 phagemid recombinant antibodies display system. PADI4 SNP was genotyped by reverse transcription cDNA sequencing and heterozygote was DNA haplotype was mapped by TA clone sequencing. HLA-DRB1 SE alleles were analyzed by sequence specific primer-polymerase chain reaction (SSP-PCR). The prevalence of synovial CCP expression was significantly different between RA group and the control (76.9% and 11.4% respectively, P < 0.01). The frequencies of 2 SNPs (PADI4_89 G+ and PADI4_104 T+) varied significantly between the groups(P < 0.05). Compared with the major haplotypes, only these two minor alleles were associated with the increased RA susceptibility (OR = 3.67 and 2.53, P < 0.05). SE+ alleles was strongly associated with RA susceptibility (OR = 5.57, P < 0.01). The synovial expression of CCP in RA was strongly associated with SE+ alleles, only 2 minor SNPs (PADI4_89 G+ and PADI4_104T+) and the combination. Serum anti-CCP titers were significantly associated with SE+ alleles, PADI4_104T+, SE+/PADI4_89 G+ and SE+/PADI4_104T+ haplotype. The synovial expression of CCP and the generation of anti-CCP antibodies are strongly associated with SE alleles and/or certain PADI4 gene SNP in RA.

The seasonal distribution of airborne fungi in two hospitals in Istanbul

Changes in fungal concentrations depend on seasonal and environmental conditions. The aim of this study was to determine the seasonal distributions of the fungal flora in Dr. Siyami Ersek Chest, Heart and Vascular Center Education and Research Hospital (SEH) and Kartal Yavuz Selim State Hospital (KYSH) in Istanbul. Samples were collected seasonally from different stations (microbiology laboratory, toilet, patient waiting saloons, hospital garden and library) in these hospitals. Distributional patterns of the isolated micro fungi were evaluated for each season. In SEH, the highest number of the fungi was isolated in summer, followed by autumn, spring and winter, respectively. Among the 257 micro fungi isolated from this hospital, the most common species were Alternaria alternata (25.8%), Cladosporium cladosporioides (21.9%), Penicillium glabrum (12.9%), Penicillium brevicompactum (7.4%) and Cladosporium herbarum (3.9%). In KYSH, the highest number of fungi was isolated in summer followed by autumn, spring and winter, respectively. Among the 221 micro fungi isolated from this hospital, the most common species were Cladosporium cladosporioides (19.5%), Alternaria alternata (15.8%), Cladosporium herbarum (11.3%) and Penicillium brevicompactum (9.0%). Keywords: Istanbul, airborne fungi, hospital flora, seasonal flora

Some chronic rhinosinusitis patients have elevated populations of fungi in their sinuses

Objectives/Hypothesis:To measure the populations of 36 fungi in the homes and sinuses of chronic rhinosinusitis (CRS) and non‐CRS patients.Study Design:Single‐blind cross‐sectional study.Methods:Populations of 36 fungi were measured in sinus samples and in the home vacuum cleaner dust of CRS (n = 73) and non‐CRS patients (n = 16) using quantitative polymerase chain reaction. Etest strips containing amphotericin B, anidulafungin, caspofungin, fluconazole, and voriconazole were used to test the susceptibility of seven potentially relevant fungi.Results:Seven fungi (Alternaria alternata, Cladosporium cladosporioides types 1 and 2, Cladosporium herbarum, Penicillium brevicompactum, Penicillium crustosum, and Penicillium chrysogenum type 2) were discovered at very high concentrations in some CRS patients. In vitro antifungal susceptibility testing of these seven fungi demonstrated species specific sensitivities. Four CRS patients with marked elevations of fungal populations in their sinus samples underwent endoscopic sinus surgery. After surgical treatment, the fungal populations were reduced by several orders of magnitude.Conclusions:Seven fungi were found in very high concentrations in the sinuses of some CRS patients. Not one of the five common antifungal agents could control all seven of these fungi based on in vitro tests.

Effect of Plant Extracts on Quality of Khasi Mandarin (Citrus reticulata Blanco) Fruits During Ambient Storage

Khasi mandarin (Citrus reticulata Blanco) fruits were treated with Azadirachta indica, Ocimum sanctum, Aloe barbedensis, carbendazim, A. barbedensis + A. indica, A. barbedensis + O. sanctum, A. barbedensis + carbendazim and control (water). Treated fruits were packed in corrugated fibre boxes and kept for a total period of 28 days at ambient storage (20 ± 2 °C and 65 ± 5% RH). Prominent pathogen associated with fruit rot was identified as Penicillium brevicompactum. The fruit quality during storage was evaluated in terms of disease severity (per cent disease index), inhibition of spore germination (%) of P. brevicompactum, decay loss (%), juice content retention (%), total soluble solids (%), acidity (%), ascorbic acid content (mg/100 g) and sensory scores. O. sanctum-treated fruits exhibited minimum decay loss (3.33%) up to a total period of 28 days which was even less than carbendazim (10%). O. sanctum was also highly effective in inhibiting spore germination of P. brevicompactum (96.5%). Overall acceptability score (7.33) was also high in O. sanctum-treated fruits as compared with other treatments at the end of the storage period.

Antimicrobial activity of chitosan films containing nisin, peptide P34, and natamycin

The antimicrobial effect of edible chitosan films containing nisin, peptide P34, and natamycin was investigated. The activity of chitosan films was tested against Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Clostridium perfringens, Lactobacillus acidophilus, Aspergillus phoenicis, and Penicillium stoloniferum. The addition of nisin and P34 significantly increased the activity against bacteria. Films containing natamycin showed similar inhibition than those with chitosan alone. Chitosan films were effective to control microbial growth in minimally processed pear. Fourier transform infrared spectra of the films were similar, indicating the absence of relevant interaction between the polymer and the antimicrobial agent. The films were also analyzed by scanning electron microscopy to visualize the surface topography. Chitosan films were rough and heterogeneous. The incorporation of antimicrobials agents to edible chitosan films or covering may be a feasible and attractive method for food biopreservation.

Purification, Characterization and Antitumor Activity of L-asparaginase from Penicillium brevicompactum NRC 829

Aim: The aims of this study were to attempt to extract, purify and characterize of Lasparaginase, an antitumor agent, from Penicillium brevicompactum NRC 829. Study Design: Testing of antitumor activity of L-asparaginase against four different tumor human cell lines.

Penicillium species present in Uruguayan salami.

The surface coverage of certain dry fermented sausages such as Italian salami by some species of Penicillium provides their characteristic flavor and other beneficial properties. One of them is the protective effect by means of a uniform film of white mold against undesirable microorganisms. The aim of this work was to identify and to isolate the fungal species present in mature Italian type of salami and to evaluate if it is possible to obtain some of them as starters. In addition, the effects of temperature (14 °C and 25 °C), water activity (a w) (0.90, 0.95 and 0.995) and 2.5 % sodium chloride (NaCl) on fungal growth were determined. Similarly, the proteolytic and lipolytic activity and the ability to produce toxic secondary metabolites were evaluated in order to characterize some possible starter strain. All species found belong to the genus Penicillium, including a performing starter as Penicillium nalgiovense and some potentially toxicogenic species. All the strains showed a higher growth rate at 25 °C. The production of extracellular proteases and lipases was significantly higher at 25 °C than at 14 °C with and without sodium chloride. Only Penicillium expansum produced patulin. On the other hand, Penicillium griseofulvum was the only species that produced ciclopiazonic acid but none of the strains produced penicillin. The species present on salami, Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum and Penicillium puberulum were unable to produce any of the evaluated toxins. These findings suggest that some fungal isolates from the surface of salami such as P. nalgiovense are potentially useful as starters in sausage manufacture.

Adaptive evolution of drug targets in producer and non-producer organisms

MPA (mycophenolic acid) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMPDH (IMP dehydrogenase). The MPA-biosynthetic cluster of Penicillium brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA resistance. Surprisingly, all members of the subgenus Penicillium contain genes encoding IMPDHs of both the A and B types, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred before and independently of the acquisition of the MPAbiosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant, whereas the IMPDHs from a non-producer are MPA-sensitive. Resistance comes with a catalytic cost: whereas P. brevicompactum IMPDH-B is >1000-fold more resistant to MPA than a typical eukaryotic IMPDH, its kcat/Km value is 0.5% of 'normal'. Curiously, IMPDH-B of Penicillium chrysogenum, which does not produce MPA, is also a very poor enzyme. The MPA-binding site is completely conserved among sensitive and resistant IMPDHs. Mutational analysis shows that the C-terminal segment is a major structural determinant of resistance. These observations suggest that the duplication of the IMPDH gene in the subgenus Penicillium was permissive for MPA production and that MPA production created a selective pressure on IMPDH evolution. Perhaps MPA production rescued IMPDH-B from deleterious genetic drift.

Kinetically Controlled Drug Resistance: HOW PENICILLIUM BREVICOMPACTUM SURVIVES MYCOPHENOLIC ACID

The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 10(3)-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate.

Joint occurrence of the bubble protein and mycophenolic acid in Penicillium brevicompactum Dierckx

Colonies of the fungus Penicillium brevicompactum Dierckx contain exudate droplets on the surface of the mycelia when grown on Murashige and Skoog medium. It was previously shown that the exudate is enriched in the bubble protein. Here, we show that the exudate bubbles are also enriched in a fluorescent non-protein molecule, which upon isolation and characterisation was determined as mycophenolic acid. Based on this discovery, mycophenolic acid from exudate bubbles may constitute an alternative source for purification of the metabolite for clinical use. .

Surface Hydrophobicity of Culture and Water Biofilm of Penicillium spp.

Fungal surface hydrophobicity is involved in several functions in fungal growth and development. Water contact angles measurement has been used as a direct and simple approach for its characterisation in solid cultures. Microsphere adhesion assay is said to be the best method to assess cell hydrophobicity of filamentous fungi. This study aimed to apply these two methods to study hydrophobicity of Penicillium expansum and Penicillium brevicompactum grown as mycelial mats in solid culture, liquid culture and water biofilms. As result, both species in solid cultures were classified as hydrophobic with contact angles ≥90º, but in liquid cultures and water biofilms showed different levels of hydrophobicity when microsphere adhesion assay was applied. In addition, was found that biofilms have specific hydrophobic hyphae which may be involved in fungal ecological functions.

A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi

BackgroundMany secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF.ResultsIn accordance with our hypothesis, heterologous expression of mpaF dramatically increased MPA resistance in a model fungus, Aspergillus nidulans, which does not produce MPA. The growth of an A. nidulans strain expressing mpaF was only marginally affected by MPA at concentrations as high as 200 μg/ml. To further substantiate the role of mpaF in MPA resistance, we searched for mpaF orthologs in six MPA producer/non-producer strains from Penicillium subgenus Penicillium. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting mpaF homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class.ConclusionsWe identified a novel variant of the IMPDH-encoding gene in six different strains from Penicillium subgenus Penicillium. The novel IMPDH variant from MPA producer P. brevicompactum was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.

Microbial synthesis of gold nanoparticles using the fungus Penicillium brevicompactum and their cytotoxic effects against mouse mayo blast cancer C2C12 cells

Microorganisms, their cell filtrates, and live biomass have been utilized for synthesizing various gold nanoparticles. The shape, size, stability as well as the purity of the bio synthesized nanoparticles become very essential for application purpose. In the present study, gold nanoparticles have been synthesized from the supernatant, live cell filtrate, and biomass of the fungus Penicillium brevicompactum. The fungus has been grown in potato dextrose broth which is also found to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification, following purification and after storing the particles for 3 months under refrigerated condition. Different characterization techniques like X-ray diffraction, Fourier transform infrared spectroscopy, and UV-visible spectroscopy have been used for analysis of the particles. The effect of reaction parameters such as pH and concentration of gold salt have also been monitored to optimize the morphology and dispersity of the synthesized gold nanoparticles. A pH range of 5 to 8 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. Additionally, the cytotoxic nature of prepared nanoparticles has been analyzed using mouse mayo blast cancer C(2)C(12) cells at different time intervals (24, 48, and 72 h) of incubation period. The cells are cultivated in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum with antibiotics (streptopenicillin) at 37°C in a 5% humidified environment of CO(2). The medium has been replenished every other day, and the cells are subcultured after reaching the confluence. The viability of the cells is analyzed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method.