Loss of FZO1 gene results in changes of cell dynamics in fission yeast.

Mitochondrial fission and fusion dynamics are critical cellular processes, and abnormalities in these processes are associated with severe human disorders, such as Beckwith­Wiedemann syndrome, neurodegenerative diseases, Charcot­Marie­Tooth disease type 6, multiple symmetric lipomatosis and microcephaly. Fuzzy onions protein 1 (Fzo1p) regulates mitochondrial outer membrane fusion. In the present study, Schizosaccharomyces pombe (S. pombe) was used to explore the effect of FZO1 gene deletion on cell dynamics in mitosis. The mitochondrial morphology results showed that the mitochondria appeared to be fragmented and tubular in wild­type cells; however, they were observed to accumulate in fzo1Δ cells. The FZO1 gene deletion was demonstrated to result in slow proliferation, sporogenesis defects, increased microtubule (MT) number and actin contraction defects in S. pombe. The FZO1 gene deletion also affected the rate of spindle elongation and phase time at the metaphase and anaphase, as well as spindle MT organization. Live­cell imaging was performed on mutant strains to observe three distinct kinetochore behaviors (normal, lagging and mis­segregation), as well as abnormal spindle breakage. The FZO1 gene deletion resulted in coenzyme and intermediate metabolite abnormalities as determined via metabolomics analysis. It was concluded that the loss of FZO1 gene resulted in deficiencies in mitochondrial dynamics, which may result in deficiencies in spindle maintenance, chromosome segregation, spindle breakage, actin contraction, and coenzyme and intermediate metabolite levels.

Structure identification, antitumor activity and mechanisms of a novel polysaccharide from Ramaria flaccida (Fr.) Quél.

It is an important aspect of current cancer research to search for effective and low-toxicity anticancer drugs and adjuvants. Polysaccharides, as immunomodulators, can improve the immune function of the body, kill tumor cells directly and prevent tumor development by increasing the resistance of the body to carcinogenic factors. The aim of the present study was to identify natural polysaccharide compounds with novel structure and antitumor activity via the separation and analysis of polysaccharide components from Ramaria flaccida (Fr.) Quél. (RF-1). In the present study, high-performance gel permeation chromatography, gas chromatography-mass spectrometry and nuclear magnetic resonance were used to identify the structure of polysaccharides from RF-1. Subsequently, the antitumor activity and mechanism of RF-1 were studied by establishing an in vivo S180 tumor model, and by using Illumina sequencing technology and enzyme-linked immunosorbent assay (ELISA). The present results revealed that the average molecular weight of RF-1 was 17,093 Da and that RF-1 was composed of the monosaccharides glucose and galactose, with a 2:1 ratio. The main chain of RF-1 consisted of (1→6, 2)-α-D-galactopyranose and (1→6, 4)- α-D-glucopyranose. One of the branched chains was linked to 4-O of the main glucose chain by (1→6)-α-D-glucopyranose and next linked by one (→4)-ß-D-glucopyranose. The other two branched chains were both linked to 2-O of the main glucose chain by one (→4)-ß-D-glucopyranose. In addition, RF-1 inhibited the growth of S180 tumors in vivo. When the concentration of RF-1 was 20 mg/kg, the inhibition rate of S180 tumors in mice was 48.4%. Compared with the blank control group, 1,971 differentially expressed genes were identified, of which 818 were upregulated and 1,153 were downregulated in the RF-1 group. A Gene Ontology enrichment analysis generated 47,091 assignments to biological processes, 5,250 assignments to cellular components, and 6,466 assignments to molecular functions. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the Wnt and MAPK signaling pathways were significantly enriched. The number of differentially annotated genes in these two pathways was 19 and 33, respectively. Additionally, ELISA results revealed that the protein levels of interleukin (IL)-1ß, IL-6, vascular endothelial growth factor (VEGF) and VEGF receptor in the RF-1 group were significantly downregulated compared with the S180 blank control group (P<0.01).

Overexpression of PTEN may increase the effect of pemetrexed on A549 cells via inhibition of the PI3K/AKT/mTOR pathway and carbohydrate metabolism.

Lung cancer is one of the leading causes of tumor­associated mortality, and >75% of patients with lung cancer have non­small cell lung cancer (NSCLC). Pemetrexed, a folate antagonist, is a first­line chemotherapy drug for NSCLC that is administered alone or in combination with cisplatin. The present study established in vitro cell models of PTEN inhibition and overexpression, and the effects of the treatment with pemetrexed were investigated in these cell models. Result from the present study demonstrated that treatment with pemetrexed suppressed lung cancer cell proliferation, inhibited mRNA and protein expression levels of anti­apoptotic Bcl2, and increased the mRNA and the protein expression levels of pro­apoptotic p53 and apoptosis regulator BAX. The present study suggested that pemetrexed regulated apoptosis via the inhibition of the mTOR/PI3K/AKT signaling pathway. Additionally, cellular processes associated with the aerobic oxidation of carbohydrates were identified to be significantly inhibited. The present findings suggested that treatment with pemetrexed may exhibit synergistic effects with PTEN on lung cancer cells via the inhibition of the PI3K/AKT/mTOR signaling pathway and through carbohydrate metabolism, and treatment with pemetrexed combined with PTEN overexpression may represent a novel therapeutic strategy for the treatment of NSCLC.

Structural characterization and immune regulation of a novel polysaccharide from Maerkang Lactarius deliciosus Gray.

The present study investigated the structural characterization and immune regulation of a novel polysaccharide from Maerkang Lactarius deliciosus Gray. Chemical methods, high performance gel permeation chromatography, fourier transform infrared spectroscopy, nuclear magnetic resonance spectrum and gas chromatography­mass spectrometry were used to characterize the polysaccharide structure. The immunomodulatory abilities of the Maerkang L. deliciosus Gray polysaccharide (LDG­M) were also investigated. LDG­M was primarily composed of ß­D­glucose and α­D­lyxose with the ratio of 2:1. The possible structure of LDG­M had a backbone of 1,6­linked­ß­D­glucose and 1,4,6­linked­ß­D­glucose, with branches primarily composed of one (1→4)­linked­α­D­lyxose residue. The immunoregulatory activity results demonstrated that LDG­M promoted the proliferation and phagocytosis of macrophages, and induced cytokine release. LDG­M also promoted the proliferation of B cells by affecting the G0/G1, S and G2/M phases. The present study introduced LDG­M as a valuable source with unique immunoregulatory properties.

Protein chip analysis of cytokines reveals a key mechanism of the antitumor and immunostimulatory activities of Tricholoma matsutake polysaccharide.

Polysaccharide has been widely used in medical and health field because of its function of immune regulation. The aim of present study was to use protein chip to test the 200 cytokines secreted by macrophages which were induced by the polysaccharides of Tricholoma matsutake (TMP-A) to study the role of TMP-A acting on macrophages and its mechanism, further understanding the mechanism of the TMP-A effect on immune activity. The results of the analysis indicated that among all of these cytokines, including IL-1ß, IL-10, IL-23, TNF-α, CD40L, G-CSF, etc. there are 73 up-regulated and 43 down-regulated cytokines. The KEGG analysis indicated that T. matsutake polysaccharide can influence the immune response of macrophages through a series of signaling pathways, and the three major signaling pathways are Jak-STAT signaling pathway, PI3K-Akt signaling pathway and NF-kappa B signaling pathway. Those three signaling pathway are closely related to the pathogenesis of many diseases. The results showed that TMP-A can activate immune cells to regulate the immunity.

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.

Structure elucidation and bioactivities of a new polysaccharide from Xiaojin Boletus speciosus Frost.

A new water-soluble polysaccharide named BSF-X was extracted and purified from the fruiting bodies of Boletus speciosus Frost which had a molecular weight of 141,309 Da. The structure identification results showed that BSF-X was mainly composed of ß-d-glucose and α-D-galactose. BSF-X had a backbone of 1, 4-linked ß-d-glucose of which branches were mainly composed of two 1, 6-linked α-D-galactose residue and a 4-linked ß-d-glucose at the end of the branches. Antitumor activities results showed that BSF-X could inhibit the proliferation of L929 cells in vitro and S180 tumor cells in vivo. Immunoregulatory activities results showed that BSF-X could promote the proliferation of T cells, B cells and macrophages by promoting the cells into S phase from G0/G1 phase. Polysaccharide BSF-X can also enhance the phagocytes is and cytokine secretion of macrophages. This study introduced the new polysaccharide BSF-X as a valuable source which exhibit unique antitumor and immunoregulatory 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.

[Tumor Associated Fibroblasts Promote PD-L1 Expression in Lung Cancer Cells].

BACKGROUND: Tumor-associated fibroblasts (TAF) is an important part of TME, which inhibits the function of immune cells. CD8+ T cells play a significant role in tumor immunity. T-cell membrane possesses a distinct type of molecule with a negative regulatory function. Upon interaction with its corresponding ligand [programmed death factor ligand 1 (PD-L1)], programmed death factor 1 (PD-1) is activated and thus inhibits the kinase activity of T cells. This study aims to explore the possible effects of TAF on PD-L1 expression in lung cancer cells. METHODS: Lung cancer cell lines H1975 and H520 were co-cultured with (experiment) or without TAF (control) via Transwell assay for through 48 hours under the same culture condition. H1975 and H520 cells were counted using a microscope. The protein and mRNA expression levels of PD-L1 were detected by FCM assay and PCR analysis, respectively. RESULTS: The numbers of lung cancer cells in 100 µm2 for H1975 and H520 cells are (46±21) and (38±10) in the experiment group, respectively, and (16±5) and (12±5) in the control group, respectively (P<0.05). The expression levels of the PD-L1 protein in H1975 and H520 cells are (20.93%±3.54%) and (19.26%±3.04%) in the experiment group, respectively, and (12.58%±2.52%) and (11.60%±2.65%) in the control group, respectively (P<0.05). The mRNA expression levels in H1975 and H520 cells are (16.45±1.25) and (15.38±2.02) pg/mL in the experiment group, respectively, and (7.78±1.27) and (7.20±1.58) pg/mL (P<0.05) in the control group, respectively (P<0.05). CONCLUSIONS: TAF promotes the growth and increases the expression of PD-L1 in H1975 and H520 cells.
.

[Altered Treg and IL-1A Expression in the Immune Microenvironment 
of Lung Squamous-cell Cancer after EGFR Blockade].

BACKGROUND: Targeting the mutations and amplifications in the epidermal growth factor receptor (EGFR) gene has curative effects on cancers of the lung, oral cavity, and gastrointestinal system. However, a systemic immune inflammation is an adverse effect of this therapeutic strategy. In this study, we aimed to identify the possible changes in the tumor microenvironment that contribute to the anti-cancer activity of EGFR inhibition. METHODS: Squamous-cell cancers were induced by the syngeneic transplantation of either EGFR-null or wild-type mouse primary keratinocytes that had been transduced with an oncogenic H-ras retrovirus. The mice were treated with gefinitib. Then, flow cytometric was used to detect the ratio of T cells and the expression of programmed cell death receptor 1 (PD-1). RT-PCR was used to detect the expression of cytokines and chemokines. RESULTS: Tumors that formed from EGFR-null keratinocytes were smaller, had fewer infiltrating FoxP3+ Treg cells, lower Foxp3 RNA, and lower percentage of PD-1 positive CD4 cells than those formed from wild-type keratinocytes. These results indicated that tumor cells can autonomously regulate the tumor microenvironment. Hosts with wild-type cancers and that were treated with gefitinib for 1 week tended to have smaller tumors. The treated mice in the short-term pharmacological model tended to have reduced FoxP3+ cells and FoxP3 RNA in the tumor microenvironment, as well as a substantially increased ratio of IL-1A/IL-1RA transcripts. These results suggested that the brief systemic inhibition of EGFR signaling alters the immune environment of the targeted cancer. CONCLUSIONS: The autonomous (genetic) or systemic (pharmacologic) inhibition of EGFR signaling in tumor cells reduces tumor growth and Treg infiltration in the tumor microenvironment. An EGFR-dependent Treg function supports the growth of squamous cancers. Therefore, Treg is a target in the therapeutic strategy of EGFR inhibition.

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.

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.