Effect on macrophage proliferation of a novel polysaccharide from Lactarius deliciosus (L. ex Fr.) Gray.

The fundamental mechanisms underlying the preventional and therapeutic effects of polysaccharides from fungi, including the immunostimulatory, antiviral and antitumor effects, are considered to occur through the modulation and stimulation of the macrophage and complement system. LDG-A, a novel polysaccharide from Lactarius deliciosus (L. ex Fr.) Gray exhibits marked antitumor activities in vivo. However, the underlying molecular mechanism of the antitumor activities of LDG-A remains unclear. In the present study, cell cycle analysis was performed in macrophages and B cells, and the transcriptomes of macrophages in the control group and LDG-A group were sequenced using Illumina sequencing technology to analyze the differentially expressed genes (DEGs), and elucidate the molecular mechanisms underlying the immunomodulatory and antitumor activities of LDG-A. The cell cycle analysis results indicated that LDG-A was able to promote the proliferation of B cells by promoting cell cycle progression in S phase and G2/M phase and eliminating cell cycle arrest in G0/G1, and promote the proliferation of macrophages by promoting cell cycle progression in G0/G1 phase and eliminating cell cycle arrest in G2/M phase. Of the total number of genes (8,140), ~77.00% were expressed [reads per kilobase per million reads (RPKM) ≥1] and 1,352 genes were highly expressed (RPKM >60) in the LDG-A group. Of 775 unigenes which were identified as DEGs, 469 were downregulated and 306 genes were upregulated. A protein chip method was also used to determine the cytokines secreted by macrophages. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and GO enrichment analysis indicated that the Janus kinase/signal transducer and activator of transcription, mitogen-activated protein kinase, chemokine, vascular endothelial growth factor and transforming growth factor ß signaling pathways are markedly enriched for DEGs.

Structural characterization, immune regulation and antioxidant activity of a new heteropolysaccharide from Cantharellus cibarius Fr.

A new heteropolysaccharide was extracted and purified from the fruiting bodies of Cantharellus cibarius Fr. The Cantharellus cibarius Fr. polysaccharide (CC-1) had a molecular weight of 61,056 kDa and was mainly formed of the glucose and xylose at ratio of 5:1. Structure identification of CC-1 was analysed by a combined application of total hydrolysis, high performance liquid chromatography (HPLC), methylation analysis, gas chromatography-mass spectrometry (GC-MS), infrared (IR) spectra and nuclear magnetic resonance (NMR) spectroscopy. The experimental results showed that CC-1 had a backbone of 1,4-linked-ß-D-glucose which branched at O-6 and the branches were mainly composed of 6→1)-α-D-xylopyranose residue. CC-1 exhibited significant in vitro antioxidant effect and proliferation effect of immune cells. The activity study showed CC-1 has ability to clear the ABTS+ free radical and DPPH- free radical in a certain range of concentration. The proliferation activity of the immune cells showed that the proliferation effect on B cells was very significant (P<0.001) in the concentration of 0.625-80 mg/ml; and the effect of T cell proliferation was also very significant (P<0.001) in the concentration of 5-20 mg/ml. The result of this study introduced Cantharellus cibarius Fr. as a possible valuable source in exhibiting unique immunoregulatory and antioxidant properties.

Comparative Analysis of Transcriptomes of Macrophage Revealing the Mechanism of the Immunoregulatory Activities of a Novel Polysaccharide Isolated from Boletus speciosus Frost.

BACKGROUND: The mechanism of the immunoregulatory activities of polysaccharide is still not clear. MATERIALS AND METHODS: Here, we performed the B-cell, T-cell, and macrophage cell proliferation, the cell cycle analysis of macrophage cells, sequenced the transcriptomes of control group macrophages, and Boletus speciosus Frost polysaccharide (BSF-1) group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) to determine the molecular mechanisms of immunomodulatory activity of BSF-1 in macrophages. RESULTS: These results suggested that BSF-1 could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell division. A total of 12,498,414 and 11,840,624 bp paired-end reads were obtained for the control group and BSF-1 group, respectively, and they corresponded to a total size of 12.5 G bp and 11.8 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 81.83% of the total number of genes (8,257) were expressed reads per kilobase per million mapped reads (RPKM ≥1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 group. A gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathways are significantly enriched for DEGs between the two cell groups. CONCLUSION: An analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of BSF-1. Based on the experimental data, we believe that the significant antitumor activities of BSF-1 in vivo mainly involve the MAPK signaling pathways. SUMMARY: Boletus speciosus Frost-1 (BSF-1) could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell divisionApproximately 81.83% of the total number of genes (8257) were expressed (reads per kilobase per million mapped reads [RPKM] =1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 groupA gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functionsA Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase signaling pathways are significantly enriched for DEGs between the two cell groups. Abbreviations used: BSF-1: Boletus speciosus Frost polysaccharide.

Structure elucidation and antitumor activity of a new polysaccharide from Maerkang Tricholoma matsutake.

A new heteropolysaccharide was isolated from the fruiting bodies of Tricholoma matsutake which had a molecular weight of 12078 Da. The results of structural features analysis showed that T. matsutake polysaccharide, here named TMP-B, was mainly composed of α - D - glucose and α - D - galactose which ratios were 7:2 and had a backbone of 1, 4 - linked α - D - glucose which branches were mainly composed of two 6 - linked α - D - galactose residue, and the α - D - galactose was 1, 6 - linked. Antitumor activity results showed that heteropolysaccharide TMP-B could inhibit the growth of S180 tumor in vivo and promote the apoptosis of L929 cells in vitro. Immunoregulatory activity results showed that TMP-B could promote the proliferation of macrophages by affecting G0/G1 phase, S phases and G2/M phases and promote cytokines release and gene expression. The result of this study introduced Maerkang T. matsutake as a possible valuable source which helped to exhibit unique antitumor and immunoregulatory properties.

Preventative effect of OMZ-SPT on lipopolysaccharide-induced acute lung injury and inflammation via nuclear factor-kappa B signaling in mice.

Acute lung injury (ALI) is an early pathophysiologic change in acute respiratory distress syndrome and its management can be challenging. Omalizumab (Xolair™) is a recombinant DNA-derived, humanized antibody. OMZ-SPT is a polypeptide on the heavy chain of omalizumab monoclonal antibody. Here, we found that intramuscular administration of OMZ-SPT significantly improved survival and attenuated lung inflammation in female C57BL/6 mice suffering from lipopolysaccharide (LPS)-induced ALI. We also demonstrated that OMZ-SPT can inhibit expression of the inflammatory cytokines tumor necrosis factor-α, interleukin-1ß and interleukin-6 by ELISA in mice suffering from LPS-induced ALI and a mouse macrophage line (RAW264.7 cells). In addition, we showed that OMZ-SPT inhibited LPS-induced activation of nuclear factor-kappa B (NF-κB) signaling and total expression of NF-κB by western blotting. These data suggest that OMZ-SPT could be a novel therapeutic choice for ALI.

Structure and antioxidant activity of a novel polysaccharide derived from Amanita caesarea.

A heteropolysaccharide was isolated from the fruiting bodies of Amanita caesarea using a diethylaminoethyl-cellulose column, Sephacryl S­300 gel column and Sephadex G­200 column. The Amanita caesarea polysaccharide was predominantly composed of α-D-glucose and α-D-lyxose at a ratio of 2:1, and it had a molecular weight of 19,329 Da. The structural features of the Amanita caesarea polysaccharide were investigated by a combination of total hydrolysis, methylation analysis, gas chromatography-mass spectrometry, and infrared spectra and nuclear magnetic resonance spectroscopy. The results showed that Amanita caesarea polysaccharide (termed AC­1) had a backbone of 1,4­linked α­D­glucose and 1,3,6­linked α­D­glucose, with branches of one 1­linked α­D­lyxose residue. The antioxidant activity of AC­1 was evaluated by two biochemical methods, 2,2-azino-bis diammonium (ABTS+) radical scavenging activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH-) radical scavenging activity. The uncontrolled production of free radicals is involved in various diseases, including cancer, atherosclerosis and degenerative aging processes. The results indicated that the Amanita caesarea polysaccharide exhibits strong antioxidant activity, thus, it may be a useful natural product antioxidant.

Structure elucidation, proliferation effect on macrophage and its mechanism of a new heteropolysaccharide from Lactarius deliciosus Gray.

A new heteropolysaccharide was isolated from the fruiting bodies of Lactarius deliciosus Gray which had a molecular weight of 16kDa and was mainly composed of the galactose and glucose. Structural elucidation results indicated that Lactarius deliciosus Gray polysaccharide (LDG-B) had a backbone of (1,6)-linked d-galactose and (1, 2, 6)-linked d-galactose which branches were mainly composed of 4-linked d-glucose and 6-linked d-galactose residue. Cell cycle test results showed that LDG-B could promote the proliferation of B cells and macrophage cells by affecting G0/G1 phase, S phases and G2/M phases. The analysis of transcriptomes sequence of macrophages showed a total of 1839 genes were identified as DEGs, and approximately 708 genes were up-regulated, whereas 1131 genes were down-regulated in LDG-B group. KEGG pathway enrichment analysis showed that the MAPK, JAK-STAT and NF-κB signaling pathways are significantly enriched for DEGs in LDG-B group. Analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of LDG-B.

Comparative analysis of macrophage transcriptomes reveals a key mechanism of the immunomodulatory activity of Tricholoma matsutake polysaccharide.

In the present study, we performed a proliferation assay, phagocytosis assay and cell cycle analysis of macrophages and sequenced the transcriptomes of control group macrophages and TMP-A group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) and determine the molecular mechanisms associated with differences in the immunomodulatory activity of TMP-A in macrophages. The results showed that TMP-A exhibits strong proliferation activity and phagocytosis activity in RAW264.7 cells in vitro and could also promote the proliferation of macrophage cells by abolishing cell-cycle arrest in the G0/G1 phase and promoting the cell cycle in the G2/M phase, which may induce cell division. A total of 12,616,096 and 11,798,839 bp paired-end reads were obtained for the control group and TMP-A group, respectively, and they corresponded to a total size of 12.5 G bp and 11.7 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 79.8% of the total number of genes (10,191) were expressed (RPKM ≥1), and more than 1,372 genes were highly expressed (RPKM >60) in the TMP-A group. A total of 1,043 unigenes were identified as DEGs, and approximately 486 genes were upregulated, whereas 557 genes were down-regulated, which might have contributed to the proliferation activity and phagocytosis activity of TMP-A in the RAW264.7 cells in vitro. A Gene Ontology (GO) enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A KEGG pathway enrichment analysis showed that the MAPK and NF-κB signaling pathways are significantly enriched for DEGs between the two cell groups. Based on the experimental data, we believe that the significant antitumor activities of TMP-A in vivo involve the MAPK and NF-κB signaling pathways because the two signaling pathways intersect.

Structure elucidation and anti-tumor activities of water-soluble oligosaccharides from Lactarius deliciosus (L. ex Fr.) Gray.

BACKGROUND: Oligosaccharides are composed of a variable number of monosaccharide units and very important in the biologically diverse of biological systems. MATERIALS AND METHODS: Crude water-soluble oligosaccharide was extracted from the fruiting bodies with water and then successively purified by DEAE-cellulose 52 and Sephadex G-100 column chromatography, yielding one major oligosaccharides fractions: LES-A. Structural features of Lactarius deliciosus (L. ex Fr.) Gray oligosaccharide (LDGO-A) were investigated by a combination of monosaccharide component analysis by thin layer chromatography, infrared spectra, nuclear magnetic resonance spectroscopy, scanning electron microscopy, and high-performance gel permeation chromatography analysis. RESULT: The results indicated that LDGO-A was composed of D-glucose and D-xylose, and the average molecular sizes was approximately 945 Da. The anti-tumor activity of LDGO-A was evaluated in vivo. The inhibitory rate in mice treated with 40 mg/kg LDGO-A can reach 40.02%, being the highest in the three doses, which may be comparable to mannatide. Histology of immune organs shows that the tissues arranged more regular and firmer, but the tumor tissue arranged looser in LDGO-A group than those in the control group. Meanwhile, there is no obvious damage to other organs, such as heart. The anti-tumor activity of the LDGO-A was usually believed to be a consequence of the stimulation of the cell-mediated immune response because it can significantly promote the lymphocyte and macrophage cells in the dose range of 100-400 µg/mL in vitro. LDGO-A also effected the expression of some housekeeping genes mRNA in S180 tumor. CONCLUSION: Accordingly, the LDGO-A might serve as an effective healthcare food and source of natural anti-tumor compounds.

Structure elucidation, anticancer and antioxidant activities of a novel polysaccharide from Gomphus clavatus Gray.

A novel heteropolysaccharide from the fruiting bodies of Gomphus clavatus Gray was isolated through Sephadex G-200 and DEAE-cellulose columns. The Gomphus clavatus Gray polysaccharide (GCG-1) was mainly composed of ß-D-glucosepyranose (ß-D-Glu) and α-D-galactopyranose (α-D-Gal) in a ratio of 3:2 and had a molecular weight of ~50,000 Da. The structure of GCG-1 was investigated by a combination of total hydrolysis, gas chromatography-mass spectrometry, methylation analysis, nuclear magnetic resonance spectroscopy and infrared spectra. The results indicated that GCG-1 had a backbone of (1 → 4)-ß-D-glucosepyranose residues with branches at O-6 and the branches consisted of two with (1 → 3)-α-D-galactopyranose residue. Antioxidation test in vitro showed that it possessed strong free radical scavenging activity, which may be comparable to vitamin C and butylated hydroxytoluene. GCG-1 also induced the apoptosis of HepG-2 cells and affected the mRNA expression of various housekeeping genes in the HepG-2 cells. The results indicated that Gomphus clavatus Gray may be an ideal sources for antioxidant and anticancer agents.

Composition and antioxidant activity of water-soluble oligosaccharides from Hericium erinaceus.

Oligosaccharide are carbohydrate molecules, comprising repeating units joined together by glycosidic bonds. In recent years, an increasing number of oligosaccharides have been reported to exhibit various biological activities, including antitumor, immune-stimulation and antioxidation effects. In the present study, crude water­soluble oligosaccharides were extracted from the fruiting bodies of Hericium erinaceus with water and then successively purified by diethylaminoethyl­cellulose 52 and Sephadex G­100 column chromatography, yielding one major oligosaccharide fraction: Hericium erinaceus oligosaccharide (HEO­A). The structural features of HEO­A were investigated by a combination of monosaccharide component analysis by thin layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, scanning electron microscopy and high­performance gel permeation chromatography. The results indicated that HEO­A was composed of D­xylose and D­glucose, and the average molecular size was ~1,877 Da. The antioxidant activity of HEO­A was evaluated using three biochemical methods to determine the scavenging activity of HEO­A on 1,1­diphenyl­2­picrylhydrazyl, hydrogen peroxide and 2,2'­azino­bis(3­ethylbenzthiazoline­6­sufonic acid) diammonium radicals. The results indicated that HEO­A may serve as an effective healthcare food and source of natural antioxidant compounds.

Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.

Nucleotide sequences of an important functional gene hnRNPA2/B1 from Ailuropoda melanoleuca and Ursus thibetanus mupinensis and its potential value in phylogenetic study.

The cDNA fragments of hnRNPA2/B1 were cloned from the giant panda and black bear using RT-PCR method, which were, respectively, 1029bp and 1026bp in length encoding 343 and 341 amino acids. Analysis indicated the cDNA cloned from the giant panda encoded variant B1 while the cDNA cloned from black bear encoded variant A2. Analyzing the hnRNPA2B1 peptide of the giant panda and black bear, 76 glycine residues and 86 glycine residues were, respectively, found, and moreover, most glycine are concentrated in the latter halves of the hnRNPA2B1 peptides. Functional sites prediction also showed many N-myristoylation sites existed in the glycine-rich domain, which is probably related to the role of telomere maintenance. From base bias and substitution analysis, we can conclude that the ORF of hnRNPA2/B1 biased G while hated C, and transition of the third site did not achieve the level of saturation. Orthology analysis indicated that both the nucleotide sequence and the deduced amino acid sequence showed high identity to other 26 hnRNPA2/B1 sequences from mammals and nonmammals reported. These sequences were used to construct phylogenetic trees employing the NJ method with 1000 bootstrap, and the obtained tree demonstrated similar topology with the classical systematics, which suggested the potential value of hnRNPA2/B1 in phylogenetic analysis. This report will be the first step to the study function of hnRNPA2/B1 in the giant panda and black bear, and will provide a scientific basis to disease surveillance, captive breeding, and conservation of the endangered species.

Pharmacological evaluation for anticancer and immune activities of a novel polysaccharide isolated from Boletus speciosus Frost.

The fungal polysaccharides have been revealed to exhibit a variety of biological activities, including antitumor, immune-stimulation and antioxidation activities. In the present study, the immune and anticancer activities of a novel polysaccharide, BSF-A, isolated from Boletus speciosus Frost was investigated. The inhibitory rate of S180 tumors in mice treated with 40 mg/kg BSF-A reached 62.449%, which was the highest rate from the three doses administered; this may be comparable to mannatide. The antitumor activity of BSF-A is commonly considered to be a consequence of the stimulation of the cell-mediated immune response, as it may significantly promote the macrophage cells in the dose range of 100-400 µg/ml in vitro. The levels of the cytokines, IL-6, IL-1ß and TNF-α, and nitric oxide, induced by BSF-A treatment at varying concentrations in the macrophage cells were similar to the levels in the cells treated with lipopolysaccharide. There was weak expression of the TNF-α, IL-6, IL-1ß and inducible nitric oxide synthase mRNA in the untreated macrophages, but this increased significantly in a dose-dependent manner in the BSF-A-treated cells. BSF-A also had a time- and dose-dependent effect on the growth inhibition of the Hep-2 cells, with the concentration of 400 µg/ml having the highest inhibitory rate. A quantitative PCR array analysis of the gene expression profiles indicated that BSF-A had anticancer activities that affected cell apoptosis in the Hep-2 cells. The results obtained in the present study indicated that the purified polysaccharide of Boletus speciosus Frost is a potential source of natural anticancer substances.

Overexpression, purification, molecular characterization and the effect on tumor growth of ribosomal protein L22 from the Giant Panda (Ailuropoda melanoleuca).

The ribosomal protein L22 (RPL22) protein belongs to the L22E family of ribosomal proteins. It is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of RPL22 of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL22 was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL22 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. The result indicated that the length of the fragment cloned is 414 bp, and it contains an open-reading frame of 387 bp encoding 128 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL22 protein is 14.74 kDa with a theoretical pI 9.21. The RPL22 gene can be really expressed in E. coli and the RPL22 protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 20.1 kDa polypeptide. The data showed that the recombinant protein RPL22 had a time- and dose-dependency on the cell growth inhibition rate. The human laryngeal carcinoma Hep-2 cells treated with 0.05-6 µg/ml of RPL22 for 24 h displayed significant cell growth inhibition (p<0.05, n=8) in assayed using MTT compared to the control (untreated) cells. The data indicate that the effect at low concentrations is better than high concentrations, and the concentration of 1.5 µg/ml has the best rate of growth inhibition of 47.70%. The inhibitory rate in mice treated with 1.5 µg/ml RPL22 protein can reach 43.75%. Histology of tumor organs shows that the tissues arranged looser in RPL22 group than those in control group. Meanwhile, there is no obvious damage to other organs, such as heart, lung and kidney. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL22 responsible for its anticancer activity.

Structure elucidation and antioxidant effect of a polysaccharide from Lactarius camphoratum (Bull.) Fr.

In this study, a heteropolysaccharide was isolated from the fruiting bodies of Lactarius camphoratum (Bull.) Fr. through DEAE-cellulose column and Sephadex G-200 column. The L. camphoratum (Bull.) Fr. polysaccharide (LC-1) had a molecular weight of 9279 Da and was mainly composed of ß-D-Glu and α-D-Gal which ratios were 2:1. Structural features of L. camphoratum (Bull.) Fr. polysaccharide (LC-1) were investigated by a combination of total hydrolysis, methylation analysis, gas chromatography-mass spectrometry (GC-MS), infrared (IR) spectra and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that L. camphoratum (Bull.) Fr. polysaccharide (LC-1) had a backbone of (1→4)-ß-D-glucopyranose residues which branches at O-6 based on the experimental results. The branches were mainly composed of one with →3)-α-D-galactopyranose residue. The antioxidant activity of LC-1 was evaluated with two biochemical methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH(-)) radical scavenging, scavenging activity of 2,2'-azino-bis(3-ethylbenzthiazoline-6-suphonic acid) diammonium (ABTS(+)) radical. The results indicated that LC-1 showed strong antioxidant.

Anti-microorganism, anti-tumor, and immune activities of a novel polysaccharide isolated from Tricholoma matsutake.

BACKGROUND: Many more fungal polysaccharides have been reported to exhibit a variety of biological activities, including anti-tumor, immunostimulation, anti-oxidation, and so on. The non-starch polysaccharides have emerged as an important class of bioactive natural products. OBJECTIVE: To investigate the anti-microorganism, anti-tumor, and immune activities of a novel polysaccharide (TMP-A) isolated from Tricholoma matsutake. MATERIALS AND METHODS: The anti-microorganism activity of purified polysaccharides (TMP-A) was evaluated by the inhibition zone diameter, the anti-tumor activity was evaluated by the S180 tumor cells that were implanted subcutaneously into the Kunming strain male mice in vivo, and the immune activity was evaluated by lymphocyte proliferation and macrophage stimulation, respectively. RESULTS: In this study, the most susceptible bacteria of TMP-A at a concentration of 20 mg/ml was Micrococcus lysodeikticus (inhibition zone diameter 24.38 ± 1.19 mm) and the TMP-A did not show any antifungal activity for the tested stains of the fungi. In addition, the inhibitory rate in mice treated with 80 mg/kg TMP-A could reach 68.422%, being the highest in the three doses, which might be comparable to mannatide. The anti-tumor activity of the TMP-A was usually believed to be a consequence of the stimulation of the cell-mediated immune response, because it could significantly promote the lymphocyte and macrophage cells in the dose range of 50-200 µg/mL and in the dose range of 100 - 400 µg/mL in vitro, respectively. DISCUSSION AND CONCLUSION: The results obtained in the present study indicate that the purification polysaccharide of Tricholoma matsutake is a potential source of natural broad-spectrum, anti-microorganism, anti-tumor, and immunomodulation.

Overexpression, purification, molecular characterization and pharmacological evaluation for anticancer activity of ribosomal protein S23 from the giant panda (Ailuropoda melanoleuca).

Ribosomal protein S23 (RPS23) is a component of the 40S small ribosomal subunit encoded by the RPS23 gene, which is specific to eukaryotes. The cDNA and genomic sequence of RPS23 were cloned from Ailuropoda melanoleuca (A. melanoleuca) using reverse transcription­polymerase chain reaction (RT-PCR) technology and touchdown PCR, respectively. The two sequences were analyzed preliminarily and the cDNA of the RPS23 gene was overexpressed in Escherichia coli (E. coli) BL21. The cDNA of RPS23 cloned from giant panda was 472 bp, and it contained an open reading frame (ORF) of 432 bp encoding 142 amino acids. The nucleotide sequence of the coding sequence showed a high degree of homology to some mammals as determined by BLAST analysis, similar to the amino acid sequence. The genomic sequence was 2,105 bp in length, with 4 exons and 3 introns. The primary structure analysis revealed that the molecular weight of the putative RPS23 protein was 15.80 kDa with a theoretical isoelectric point (pI) of 11.23. The molecular weight of the recombinant protein RPS23 was 21.5 kDa with a theoretical pI of 10.57. Topology prediction showed that there are seven different patterns of functional sites in the RPS23 protein of giant panda. RPS23 was successfully expressed in E. coli and its protein fused with the N­terminal His­tagged protein triggered the accumulation of an expected 21.5­kDa polypeptide. The inhibitory rate of tumor growth in mice treated with 0.1 µg/ml RPS23 protein was 49.45%, the highest in the three doses used, which may be comparable to mannatide treatment. Histology of immune organs showed that the tissues were characterized by a regular and tight arrangement, while tumor tissues of the mice in the RPS23 group exhibited a loose arrangement compared to the control group. However, there was no obvious damage to other organs, such as the heart, lung and kidney. Investigations are currently being conducted to determine the bioactive principles of the recombinant protein RPS23 responsible for its anticancer activity.

Molecular characterization of a gene POLR2H encoded an essential subunit for RNA polymerase II from the Giant Panda (Ailuropoda Melanoleuca).

The Giant Panda is an endangered and valuable gene pool in genetic, its important functional gene POLR2H encodes an essential shared peptide H of RNA polymerases. The genomic DNA and cDNA sequences were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) adopting touchdown-PCR and reverse transcription polymerase chain reaction (RT-PCR), respectively. The length of the genomic sequence of the Giant Panda is 3,285 bp, including five exons and four introns. The cDNA fragment cloned is 509 bp in length, containing an open reading frame of 453 bp encoding 150 amino acids. Alignment analysis indicated that both the cDNA and its deduced amino acid sequence were highly conserved. Protein structure prediction showed that there was one protein kinase C phosphorylation site, four casein kinase II phosphorylation sites and one amidation site in the POLR2H protein, further shaping advanced protein structure. The cDNA cloned was expressed in Escherichia coli, which indicated that POLR2H fusion with the N-terminally His-tagged form brought about the accumulation of an expected 20.5 kDa polypeptide in line with the predicted protein. On the basis of what has already been achieved in this study, further deep-in research will be conducted, which has great value in theory and practical significance.

Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca).

Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 µg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.