ABSTRACT
Background: The dried sclerotium of medicinal fungus Polyporus umbellatus (Pers.) Fries has many pharmacological functions such as diuretic and anticancer activity, in which high-content polysaccharides may play an important role. However, RNA isolation is difficult in filamentous fungi and lacking in P. umbellatus. Results: Five methods for RNA extraction from five strains collected from four provinces were assessed for their ability to recover a high-quality RNA applicable for sequence-related amplification polymorphism (SRAP) PCR and GDP-D-mannose pyrophosphorylase (GMP) gene expression profiles. Both A260/A280 and A260/A230 ratios of the best Trizol Plus + RNAiso-mate for Plant Tissue method are around 2 with a yield of 1122.00 +/- 0.21 ng ul-1. The Trizol method also showed good quality with the yield 469.60 ng ul-1. The SRAP PCR amplified clear and polymorphic bands in all five cDNA samples transcribed from RNA by using primer Me4-Em4. GMP gene fragment (1251 bp) was successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. Conclusion: All these results showed that the total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications.
Subject(s)
RNA, Fungal/isolation & purification , Polyporus/genetics , Polyporus/chemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Agaricus blazei Murill is a native Brazilian mushroom which functions primarily as an anticancer substance in transplanted mouse tumors. However, the mechanism underlying this function of A. blazei Murill remains obscure. The present study was carried out to investigate the effect of fraction FA-2-b-ß, an RNA-protein complex isolated from A. blazei Murill, on human leukemia HL-60 cells in vitro. Typical apoptotic characteristics were determined by morphological methods using DNA agarose gel electrophoresis and flow cytometry. The growth suppressive effect of fraction FA-2-b-ß on HL-60 cells in vitro occurred in a dose- (5-80 mug/mL) and time-dependent (24-96 h) manner. The proliferation of HL-60 cells (1 x 10(5) cells/mL) treated with 40 mug/mL of fraction FA-2-b-ß for 24-96 h and with 5-80 mug/mL for 96 h resulted in inhibitory rates ranging from 8 to 54.5 percent, and from 4.9 to 86.3 percent, respectively. Both telomerase activity determined by TRAP-ELISA and mRNA expression of the caspase-3 gene detected by RT-PCR were increased in HL-60 cells during fraction FA-2-b-ß treatment. The rate of apoptosis correlated negatively with the decrease of telomerase activity (r = 0.926, P < 0.05), but correlated positively with caspase-3 mRNA expression (r = 0.926, P < 0.05). These data show that fraction FA-2-b-ß can induce HL-60 cell apoptosis and that the combined effect of down-regulation of telomerase activity and up-regulation of mRNA expression of the caspase-3 gene could be the primary mechanism of induction of apoptosis. These findings provide strong evidence that fraction FA-2-b-ß could be of interest for the clinical treatment of acute leukemia.
Subject(s)
Humans , Agaricus/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , RNA, Fungal/chemistry , RNA-Binding Proteins/pharmacology , Antineoplastic Agents/isolation & purification , /analysis , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Electrophoresis, Agar Gel , /drug effects , Microscopy, Electron, Transmission , Reverse Transcriptase Polymerase Chain Reaction , RNA, Fungal/isolation & purification , RNA, Messenger/chemistry , RNA-Binding Proteins/isolation & purification , Time Factors , Telomerase/analysisABSTRACT
Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. Several properties of this fungus have been investigated so far. However, a few studies were carried out in the field of molecular biology of this fungus. In the present study, we tried to identify the subunit G of its vacuolar ATPase [V-ATPase]. Pairs of 21 nt primers were designed from highly conserved regions of the V-ATPase subunit G genes in other fungi. Mentioned primers were utilized in PCR using isolated genomic DNA template as well as cytoplasmic RNA of T.rubrum and the PCR and RT-PCR fragments were then sequenced. About 469 nucleotides were sequenced which encoded a polypeptide with 119 amino acids. Nucleotide sequence comparison in gene data banks [NCBI, NIH] for both the DNA and its deduced amino acid sequence revealed significant homology with V-ATPase subunit G genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 84% identical to the sequence of V-ATPase subunit G from other fungi. In summary, we have cloned the first V-ATPase subunit G of dermatophytes and characterized it as a member of this gene family in other eukaryotic cells