Seven compounds were isolated from fermentation extract of cave-derived Metarhizium anisopliae NHC-M3-2 by silica gel, semi-preparative HPLC and other chromatographic methods. Their structures were elucidated by UV, IR, MS and NMR methods as 2,3-dehydroindigotide G (1), (-)-regiolone (2), naphtho-γ-pyrone (3), indigotide G (4), indigotide B (5) destruxin A (6) and destruxin B (7). Compound 1 is a new glycoside naphthopyranone compound. The anti-hepatitis B virus (HBV) activity of these compounds was evaluated. The EC₅₀ and CC₅₀ of compound 3 against HBV were 4.5 μmol·L^-1 and 92.3 μmol·L^-1, respectively. This is the first report of the antiviral activity of compound 3.

BACKGROUNDRenal transplantation in sensitized candidates remains a highly significant challenge worldwide. The production of panel reactive antibody (PRA) against human leukocyte antigen (HLA) is a major risk factor in presensitized recipients. The aim of this study was to evaluate the impact of HLA matching and recipients' PRA on two-year outcome in presensitized renal allograft recipients.

METHODSWe determined the percentage of panel reactivity and specificity of anti-HLA immunoglobulin (Ig) G antibodies in 73 presensitized renal allograft recipients compared with 81 unsensitized recipients (control group). HLA genotyping of both recipients and corresponding donors was performed by PCR with sequence-specific primers (PCR-SSP). We analyzed the factors influencing the early graft outcome (two-year rejection rates and survival rates of the grafts), including HLA mismatching, class and degree of panel reactivity, and target antigen of donors.

RESULTSPresensitized recipients had a worse two-year outcome than unsensitized recipients (P = 0.019 for rejection rate, P = 0.01 for survival rate). The difference in number of HLA-mismatched alleles with either 6-antigen matching (Ag M) standard or amino acid residue matching (Res M) standard was not significant between the rejection and non-rejection groups of presensitized recipients or between the graft survival group and graft loss group. Compared with the control group, recipients with both PRA-I and PRA-II antibodies had a significantly worse two-year outcome (P = 0.001 for rejection rate, P = 0.002 for survival rate). The two-year outcomes of the peak PRA >/= 50% group and its subgroup, at-transplant PRA > or = 50% group, were significantly worse compared with the control group (P = 0.025 and P = 0.001 for rejection rate, P = 0.043 and P = 0.024 for survival rate). The rejection rates of the at-transplant target antigen positive group and its subgroup, HLA-I target antigen positive group, were significantly higher than the control group (P = 0.001 and P = 0.001), target antigen negative group (P = 0.003 and P = 0.001), and peak target antigen positive with negative at-transplant target antigen group (P = 0.024 and P = 0.002). Two-year graft survival rates of the target antigen positive group and HLA-I target antigen positive group were significantly lower than the control group (P = 0.012 and P = 0.001). The two-year outcome of target antigen unknown group was similar to that of the target antigen positive group. Presensitized recipients with pre-transplant plasmapheresis or immunoadsorption (PRA prepared group) had a better but non-significant two-year outcome than the control group. However, the PRA unprepared presensitized recipients were different to the control group (P = 0.004 for rejection rate and P = 0.005 for survival rate). Hyperacute rejection (HR) occurred in three recipients with positive HLA-I target antigen and without mismatch according to Res M and in one case with positive PRA-II (for an unknown target antigen). No HR occurred in eight cases with positive HLA-II target antigens.

CONCLUSIONSPre-transplant PRA preparations might improve the access of presensitized patients to renal donors. Avoiding antigen-positive donors remains a fundamental measure in preventing HR and early rejections.

Adult ; Enzyme-Linked Immunosorbent Assay ; Female ; Graft Rejection ; immunology ; Graft Survival ; immunology ; HLA Antigens ; immunology ; Histocompatibility Testing ; Humans ; Isoantibodies ; blood ; Kidney Transplantation ; adverse effects ; immunology ; mortality ; Male ; Middle Aged ; Transplantation, Homologous ; immunology ; Treatment Outcome

To investigate the relationship and significance of Wnt/b-catenin signaling pathway with caspase-3, XIAP, HSP27and Grp-78. The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against b-catenin. After 72 and 96 h, protein was extracted and the protein expressions of b-catenin, caspase-3, XIAP, Grp-78 and HSP27 were detected by Western blot. b-catenin protein expression was inhibited at both time points and the expression at 96 h was higher than that at 72 h (F = 160.72, P is less than to 0.01). Interestingly, Caspase-3 protein expression was decreased at 72 h and increased to normal at 96 h (F = 136.10, P is less than to 0.01), while p-caspase-3 protein expression increased at 72 h and decreased to normal at 96 h (F = 98.65, P is less than to 0.01). XIAP protein expression decreased at 72 h (F = 37.29, P is less than to 0.01) and increased at 96 h. Grp-78 protein expression increased at 72 h and decreased to normal at 96 h ( F = 58.72, P is less than to 0.01). HSP27 protein expression showed no change following transfection ( F = 1.91, P is more than to 0.05). Wnt/b-catenin signaling pathway is related to the protein expressions of caspase-3, XIAP and Grp-78, but not related to HSP27 protein expression in HCC. Wnt/b-catenin signaling pathway may participate in the regulation of HCC apoptosis, proliferation and differentiation through affecting these factors.
Carcinoma, Hepatocellular ; Caspase 3 ; Catenins ; Humans ; Liver Neoplasms ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo explore whether the nerve growth factor has protective effects on PC12 cells from injury induced by 2, 5-hexanedione.

METHODSWith PC12 cells as the model of neurons, different concentrations of NGF were added into the culture of PC12 cells. Then cell viability was tested with MTT. The DNA fragment was observed with agarose gel electrophoresis. The apoptosis ratio was tested with flow cytometry (FACS). The p53 protein was detected with western blot. The differences among the groups were compared.

RESULTSCell viabilities were increased with the increase of the concentrations of NGF (P < 0.05). The DNA fragment, the apoptosis ratio and the expression of p53 were all decreased with the increase of the concentrations of NGF (P < 0.05).

CONCLUSIONThe NGF might have direct nutritional effects on PC12 cells, and protect them from injury induced by 2, 5 HD. Moreover, it might also have anti-apoptosis effect to some extent.

Animals ; Apoptosis ; drug effects ; DNA Fragmentation ; drug effects ; Dose-Response Relationship, Drug ; Electrophoresis, Agar Gel ; Flow Cytometry ; Hexanones ; toxicity ; Mice ; Nerve Growth Factors ; pharmacology ; PC12 Cells ; Rats ; Tumor Suppressor Protein p53 ; biosynthesis

This study was to investigate the differential regulation of CCR5 expression on T cells in healthy donors after mobilization with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and analyze its correlation with acute graft-versus-host disease (aGVHD) so as to understand the possible mechanisms underlying rhG-CSF-induced immune tolerance. Sixty-eight related healthy donor and their corresponding recipient for allogeneic hematopoietic stem cell transplantation (allo-HSCT) were enrolled in this study. The expression of CCR5 on CD4(+) and CD8(+) T cells in the peripheral blood (PB) before and after mobilization were detected by using flow cytometry (FCM) respectively. According to the changes of CCR5 expression on CD4(+) and CD8(+) T cells, the Sixty-two evaluable donors were divided into the downregulated and unchanged/upregulated (non-downregulated) groups, and the incidence of grades II to IV aGVHD in two groups were compared. The results showed that the mean value of CCR5 expression on CD4(+) and CD8(+) T cells in PB was not different significantly after mobilization (P > 0.05). Apparent inconsistency was showed among different individuals. Thirty-four (50%) donors displayed downregulation of CCR5 expression, while 34 (50%) donors manifested unchanged or upregulated CCR5 expression on CD4(+) T cells. CCR5 expression on CD8(+) T cells was downregulated in 42 (61.8%), unchanged or upregulated in 26 (38.3%) donors. The cumulative incidence of grades II to IV aGVHD in the downregulated and non-downregulated groups for CD4(+) T cells were 16.1% and 41.9% (P = 0.032), and recipients with CCR5 downregulation on CD8(+) T cells showed an increased tendency of developing aGVHD (37.8% vs 16.0%, P = 0.065). In conclusion, rhG-CSF mobilization could lead to differential regulation of CCR5 expression on T cells, which might influence the migration of T cells in vivo, decrease T cell trafficking towards GVHD target organs, and thus reduce the incidence of aGVHD after transplantation.
Adolescent ; Adult ; Blood Donors ; Child ; Child, Preschool ; Female ; Gene Expression Regulation ; Graft vs Host Disease ; pathology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; Receptors, CCR5 ; metabolism ; T-Lymphocytes ; drug effects ; metabolism ; Young Adult

OBJECTIVETo investigate the reverse effect of all-trans retinoic acid (ATRA) on Benzo (a) pyrene (B (a) P)-induced cyclin D1, CDK4, E2F-1 and E2F-4 expression and cell cycle progression in human embryo lung fibroblast (HELF).

METHODSAfter HELF cells was treated with ATRA, they were exposed to 2 micromol/L of B (a) P. Western blotting was employed to detect protein expression level; the RNA transfection techniques was used to investigate ATRA-induced signal pathway; flow cytometry was used to detect cell cycle progression.

RESULTAfter treatment with 2 micromol/L B (a) P for 24 h, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF; the expression of E2F-4 and CDK4 were not changed markedly; pretreatment with 0.1 micromol/L ATRA for 24 h could efficiently decrease B (a) P-induced overexpression of cyclin D1 and E2F-1; stimulation to antisense cyclin D1 or antisense CDK4 by B (a) P could significantly impair E2F-1 up-regulation; pretreatment with ATRA, cells with antisense cyclin D1 or antisense CDK4 showed a less decrease in B (a) P-induced overexpression of E2F-1 compared to similarly treated control cells; flow cytometry analysis showed B (a) P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B (a) P-induced cell cycle progression by an accumulation of cells in the G(1) phase.

CONCLUSIONATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Lung ; cytology ; metabolism ; Signal Transduction ; drug effects ; Tretinoin ; pharmacology

OBJECTIVETo study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.

METHODSThe phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.

RESULTSThe phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.

CONCLUSIONERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; embryology ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo observe the therapeutic effect of digital acupoint pressure for treatment of the nerve root type of cervical spondylosis.

METHODSFour hundred cases were randomly divided into a digital acupoint pressure group (DAP group) and a medicine group, 200 cases in each group. Acupoints of Quepen (ST 12), Jianjing (GB 21) and Tianzong (ST 11) etc. were used for digital pressing in the DAP group; and Chinese herb medicine of Gentongping was routinely taken in the medicine group. After three treatment courses, the symptoms of pain and numbness, the signs of pressure measurement by compression of head, brachial plexus drawer test and arm myodynamia, as well as the total cumulative scores of daily living capability, were compared.

RESULTSAfter treatment, the total cumulative scores of numbness, pressure measurement by compression of head, brachial plexus drawer test, arm myodynamia and daily living capability in both groups were obviously better than those of before treatment (all P<0.01); but there was a significant difference on the total cumulative score of the symptoms and signs between the two groups. The cured rate of 78.0% and total effective rate of 99.0% in the DAP group were better than those of 61.0% and 87.0% in the medicine group, respectively (both P<0.01).

CONCLUSIONDigital acupoint pressure plays an active role in improving the symptoms and signs on patients with nerve root type of cervical spondylosis, which is better than Chinese herb medicine of Gentongping.

Acupuncture Points ; Adolescent ; Adult ; Aged ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Hypesthesia ; drug therapy ; physiopathology ; therapy ; Male ; Massage ; Middle Aged ; Spondylosis ; drug therapy ; physiopathology ; therapy ; Young Adult

OBJECTIVETo study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELF) cells.

METHODSThe stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle.

RESULTSB[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C (10, 100, 500, 1000, and 5000 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone.

CONCLUSIONSB[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.

Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; Blotting, Western ; methods ; Cell Cycle ; drug effects ; physiology ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; G1 Phase ; drug effects ; physiology ; Humans ; Lung ; cytology ; embryology ; RNA, Antisense ; genetics ; S Phase ; drug effects ; physiology ; Transfection ; methods

To investigate the effect of hepatitis B virus X protein(HBx) on CtBP-interacting protein(CtIP) which is an important repair factor of DNA double strand break damage in HepG2 cells induced by bleomycin. A HBx stably expressing HepG2 cell line and a control HepG2 cell line with empty vector transfected were established. After the double strand break (DSB) damage occurred, the mRNA and protein levels of CtIP were detected by Real-time PCR and Western blot assay respectively, cell cycle profiles and apoptotic cell death were determined by a flow cytometry, and the position of CtIP in cells was observed by confocal laser scanning microscopy. It showed that HepG2 cells transfected with hepatitis B virus X gene could stably express HBx protein. After being induced by bleomycin, the percentage of apoptotic cell was 16.90%+/-0.89% in HBx stably expressing HepG2 cell line and 15.30%+/-0.86% in control cell line, respectively (q = 2.074, P is more than to 0.05). While the percentage of death cell was 8.71%+/-0.74% in HBx stably expressing HepG2 cell line and 4.90%+/-0.46% in control cell line, respectively (q = 7.126, P is less than to 0.01). The two cell lines manifested the increase of G2/M arrest and significant difference existed between the two cell lines. HBx down regulated the expression levels of CtIP and its mRNA. The CtIP level was 0.66+/-0.04 in HepG2-HBx cell and 0.73+/-0.05 in HepG2-vec cell, respectively (t = 2.314, P is less than to 0.05). The relative mRNA level was 1.00+/-0.06 in HepG2-HBx cell and 1.23+/-0.08 in HepG2-vec cell, respectively (t = 2. 732, P is less than to 0.05). We also found that CtIP was concentrated in the cell nucleus. The research suggests that HBx may affect DNA-repair pathways by disrupting the expression of CtIP.
Carcinoma, Hepatocellular ; metabolism ; Hep G2 Cells ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; metabolism ; Real-Time Polymerase Chain Reaction