黑盖地花菌中一个新的grifolin衍生物

从黑盖地花菌（Albatrellus yasuda）中分离得到4个grifolin衍生物：grifolene（1）,grifolin（2）,grifolicacid（3）和neogrifolin（4）,其中1为新化合物,它的化学结构通过波谱学方法鉴定。     

Neogrifolin derivatives possessing anti-oxidative activity from the mushroom Albatrellus ovinus

Three neogrifolin derivatives, 3-hydroxyneogrifolin, 1-formylneogrifolin and 1-formyl-3-hydroxyneogrifolin along with grifolin and neogrifolin were isolated from the Japanese mushroom Albatrellus ovinus belonging to Scutigeraceae. Their structures were established by a combination of two-dimensional NMR spectroscopic analyses and by chemical synthesis. 3-Hydroxyneogrifolin and 1-formyl-3-hydroxy-neogrifolin showed more potent antioxidative activity properties than either alpha-tocopherol or BHA.     

Isolation of three new meroterpenoids and seven known compounds from Albatrellus yasudae and their Aβ-aggregation inhibitory activity

Alzheimer’s disease is a serious neurologic disorder that cannot be cured completely. In this study, we targeted compounds that inhibit amyloid-beta (Aβ) aggregation, based on the amyloid cascade hypothesis. Ten compounds (1–10) were isolated from CHCl₃ extracts of the mushroom Albatrellus yasudae using Aβ-aggregation inhibitory activity–guided separation. The structures of these compounds were elucidated from 1D and 2D NMR and MS spectral data. Compounds 1–3 were novel, whereas 4–10 were identified as the known compounds grifolin, grifolic acid, neogrifolin, confluentin, 2-hydroxyneogrifolin, daurichromenic acid, and a cerebroside derivative. Compounds 1–10 were tested for Aβ-aggregation inhibitory activity. Compounds 1, 3, 5, 6, 8, and 9 have potential as Aβ-aggregation inhibitory activity.     

Grifolin, a potential antitumor natural product from the mushroom Albatrellus confluens, inhibits tumor cell growth by inducing apoptosis in vitro

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.     

A new meroterpenoid pigment from the basidiomycete Albatrellus confluens

A new farnesyl phenol named (+)-(R)-grifolinone C (1) has been isolated from the fruiting bodies of the basidiomycete Albatrellus confluens. (+)-(R)-Grifolinone C (1), a dimeric meroterpenoid, is accompanied by albatrellin (2), grifolinone B (3), grifolin (4), and grifolinone A (5). Albatrellin (2) exhibited cytotoxic activity against HepG2 human lung carcinoma cells with IC(50) value of 1.55 mug ml(-1). The structures were established on the basis of spectral evidence (IR, 1D- and 2D-NMR, and MS analyses).     

Activity in vitro and in vivo against plant pathogenic fungi of grifolin isolated from the basidiomycete Albatrellus dispansus

In the course of screening for novel naturally occurring fungicides from mushrooms in Yunnan province, China, the ethanol extract of the fruiting bodies of Albatrellus dispansus was found to show antifungal activity against plant pathogenic fungi. The active compound was isolated from the fruiting bodies of A. dispansus by bioassay-guided fractionation of the extract and identified as grifolin by IR, 1H and 13C NMR and mass spectral analysis. Its antifungal activities were evaluated in vitro against 9 plant pathogenic fungi and in vivo against the plant disease of Erysiphe graminis. In vitro, Sclerotinina sclerotiorum and Fusarium graminearum were the most sensitive fungi to grifolin, and their mycelial growth inhibition were 86.4 and 80.9% at 304.9 microM, respectively. Spore germination of F. graminearum, Gloeosporium fructigenum and Pyricularia oryzae was almost completely inhibited by 38.1microM grifolin. In vivo, the curative effect of grifolin against E. graminis was 65.5% at 304.9 microM after 8 days.     

Cytotoxic Grifolin Derivatives Isolated from the Wild Mushroom Boletus pseudocalopus (Basidiomycetes)

Activity‐guided purification of a MeOH extract of the Korean wild mushroom Boletus pseudocalopus afforded three new grifolin derivatives, 1 – 3 , along with four known phenolic compounds 4 – 7 . Their structures were established by a combination of ¹H‐ and ¹³C‐NMR, NOESY, and extensive two‐dimensional NMR spectroscopic experiments such as gCOSY, gHSQC, gHMBC, and ROESY. The major metabolites 4 and 5 were subjected to reduction to provide the side chain‐reduced compounds 8 and 9 for biological testing. All of the compounds except compound 6 showed anticancer activities in the range of IC₅₀ 3.5–11.0 μg/ml against human lung carcinoma A549 and mouse melanoma B16F1 cell lines. In addition, all compounds showed moderate radical‐scavenging activities determined by DPPH assay.     

Induction of B1 receptors in streptozotocin diabetic rats: possible involvement in the control of hyperglycemia-induced glomerular Erk 1 and 2 phosphorylation

We investigated the effects of a 3-week treatment with various combinations of angiotensin-converting enzyme inhibitor (ACEI) and B1 and B2 bradykinin receptor (B1R and B2R) antagonists (B1A and B2A) and AT1 receptor antagonist on ERK 1 and 2 phosphorylation in isolated glomeruli from streptozotocin-treated diabetic rats (STZ rats). Body weight, glycemia, and blood pressure were monitored. The rats were divided into nine groups: (1) control; and groups 2-9 were STZ treated with (3) insulin, (4) ACEI, (5) ACEI + B1A, (6) ACEI + B2A, (7) B2A, (8) B1A, (9) AT1 antagonist. ERK 1 and 2 phosphorylation and expression of B1R and B2R were assessed by Western blot analysis. ERK 1 and 2 phosphorylation was higher in STZ rats; this activation was normalized by insulin and reduced by ACEI but not by AT1 antagonist. The reduction of ERK 1 and 2 phosphorylation by the ACEI was reversed by B1A and B2A. The induction of B1R was confirmed by increased expression of mRNA and B1 receptor protein. Since ERK 1 and 2 phosphorylation is an early event in the induction of matrix secretion and hyperproliferation associated with diabetic nephropathy, activation of B1R and B2R appears to be a useful pharmacological target in the management of this pathology.     

Concept formation based on the relations among values of multi-component figures

A complex discrimination procedure was used to test class formation with multi-component figures in college students. First, selections of a red-+45 degrees -oriented rectangle (A1B1) instead of a red -45 degrees -oriented rectangle (A1B2) and of a green -45 degrees -oriented rectangle (A2B2) instead of a green-+45 degrees -oriented rectangle (A2B1), were reinforced. Second, selections of a +45 degrees -obtuse-angle-white hexagon (B1C1), instead of a +45 degrees -two-acute-angle hexagon (B1C2) and of a -45 degrees -two-acute-angle hexagon (B2C2), instead of a -45 degrees -obtuse-angle hexagon (B2C1), were reinforced. Subsequent tests with figures A1B1 and A2B1, A2B2 and A1B2, B1C1 and B2C1, and B2C2 and B1C2 demonstrated selection of the same figures as in training. Other tests with a novel figure compounded by color A1 and form C1 and a novel figure compounded by color A1 and form C2, or a novel figure compounded by A2 and C1 and a novel figure compounded by A2 and C2 demonstrated consistent selection of figures A1C1 and A2C2. A final test with figures A1C1 and A2C1, or with figures A2C2 and A1C2 also showed consistent selections of the same figures -A1C1 and A2C2. The resulting classification among selected and non-selected figures cannot be based on particular properties of the selected figures, or in a combination of a particular set of those properties, because both selected and non-selected figures were compounded by identical values. Instead, the participants selected the figures with two values of the same set (A1,B1, or C1, or A2,B2 or C2). Thus, these results demonstrated categorization based on the relations among the values of the figures. Therefore, these results have important implications for the study of concept formation.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

Reversal of Small,Dense LDL Subclass Phenotype by Weight Loss Is Associated With Impaired Fat Oxidation

Adiposity is more prevalent among individuals with a predominance of small, dense low‐density lipoprotein (LDL) (pattern B) particles than among those with larger LDL (pattern A). We tested for differences in resting energy expenditure (REE) and respiratory quotient (RQ) in overweight men with pattern A (n = 36) or pattern B (n = 60). Men consumed a standardized isoenergetic diet for 3 weeks after which a ～9 kg weight loss was induced by caloric deficit for 9 weeks, followed by 4 weeks of weight stabilization. REE and RQ were measured by indirect calorimetry before and after weight loss. Results were analyzed separately in pattern B men who converted to pattern A (B→A; n = 35) and those who did not (B→B; n = 25). At baseline, B→B men had higher trunk fat, triacylglycerol (TG) and insulin concentrations, homeostasis model assessment of insulin resistance (HOMA_IR), and smaller LDL particles compared to B→A men and baseline pattern A men who remained pattern A (A→A; n = 35). REE normalized to fat‐free mass did not change after weight loss. RQ decreased in A→A men, increased in B→A men, and did not change significantly in B→B men after weight loss. Calculated fat oxidation rates paralleled the RQ results. Baseline plasma TG concentrations were positively correlated with RQ and inversely correlated with the magnitude of weight loss achieved for a given prescribed energy reduction in the entire study population. Pattern B men who converted to pattern A with weight loss may have an underlying impairment in fat oxidation that predisposes to both dyslipidemia and an impaired ability to achieve weight loss by energy restriction.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

High-uptake and low-uptake forms of proteoglycans secreted by arterial smooth muscle cells

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans, designated as proteoglycan A and B, into the culture medium. They are isolated as immunologically distinct monomers with relative molecular masses of 280 X 10(3) and 180 X 10(3) and are characterized as chondroitin-sulfate-rich (A) and dermatan-sulfate-rich (B) proteoglycans. Both proteoglycan A and B were labelled with [35S]sulfate and used for studies of endocytosis. Uptake of proteoglycan B by arterial smooth muscle cells shows saturable kinetics. At saturation (500 microM) one cell may endocytose up to 1.5 X 10(6) proteoglycan B molecules/h. Proteoglycan A is internalized at a 10-fold lower rate. No saturation kinetics were observed at high proteoglycan A concentrations (500 microM). Endocytosis of proteoglycan B in the presence of an excess of proteoglycan A and vice versa suggest that proteoglycan A and B do not compete for the same receptor site. Free hyaluronate or chondroitin sulfate do not inhibit the uptake of proteoglycan B or A. The results suggest that proteoglycan B is internalized by arterial smooth muscle cells via a high-affinity receptor-mediated process, whereas proteoglycan A is taken up by fluid endocytosis and/or by low-affinity endocytotic processes.     

Soluble forms of the subgroup A avian leukosis virus [ALV(A)] receptor Tva significantly inhibit ALV(A) infection in vitro and in vivo

The interactions between the subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and soluble forms of the ALV(A) receptor Tva were analyzed both in vitro and in vivo by quantitating the ability of the soluble Tva proteins to inhibit ALV(A) entry into susceptible cells. Two soluble Tva proteins were tested: the 83-amino-acid Tva extracellular region fused to two epitope tags (sTva) or fused to the constant region of the mouse immunoglobulin G heavy chain (sTva-mIgG). Replication-competent ALV-based retroviral vectors with subgroup B or C env were used to deliver and express the two soluble tv-a (stva) genes in avian cells. In vitro, chicken embryo fibroblasts or DF-1 cells expressing sTva or sTva-mIgG proteins were much more resistant to infection by ALV(A) ( approximately 200-fold) than were control cells infected by only the vector. The antiviral effect was specific for ALV(A), which is consistent with a receptor interference mechanism. The antiviral effect of sTva-mIgG was positively correlated with the amount of sTva-mIgG protein. In vivo, the stva genes were delivered and expressed in line 0 chicken embryos by the ALV(B)-based vector RCASBP(B). Viremic chickens expressed relatively high levels of stva and stva-mIgG RNA in a broad range of tissues. High levels of sTva-mIgG protein were detected in the sera of chickens infected with RCASBP(B)stva-mIgG. Viremic chickens infected with RCASBP(B) alone, RCASBP(B)stva, or RCASBP(B)stva-mIgG were challenged separately with ALV(A) and ALV(C). Both sTva and sTva-mIgG significantly inhibited infection by ALV(A) (95 and 100% respectively) but had no measurable effect on ALV(C) infection. The results of this study indicate that a soluble receptor can effectively block infection of at least some retroviruses and demonstrates the utility of the ALV experimental system in characterizing the mechanism(s) of viral entry.     

Examination of molar-based distribution of A,B and C chains of amylopectin by fluorescent labeling with 2-aminopyridine

A method for determination of a molar-based distribution of A, B and C chains of amylopectin was developed. Labeling with fluorescent 2-aminopyridine was proportional to the number-average degree of polymerization (dp(n)) of the chains in the range of 6-440. Number-average chain lengths (cl(n)) of amylopectins from six different plant sources (rice, maize, wheat, potato, sweet potato and yam) determined by the labeling method were in good agreement with values obtained by determination of non-reducing residues. The molar-based distributions were polymodal (A, B(1) and B(2)+B(3) fractions) and characteristic to botanical sources. Amylopectins from starches with A-crystalline type had higher amount of A+B(1) chains (90-93% by mole) than starches with B-type (68-87%). Molar ratios of (A+B(1))/(B(2)+B(3)) were 8.9-12.9 for the A-type starches and 2.1-6.5 for the B-type starches, suggesting that amylopectins of A-type starches had 1.5-2 times more branches per cluster than B-type. The distributions of C chains, except for amylomaize, showed a broad, asymmetrical profile from dp approximately 10 to approximately 130 with a peak at dp approximately 40 and were very similar among botanical sources, suggesting that the biosynthetic process for C chains is similar in different plant species.     

Differential response to benzo[A]pyrene in human lung adenocarcinoma cell lines: the absence of aryl hydrocarbon receptor activation.

Benzo[a]pyrene (B[a]P) has been shown to produce DNA adducts and to initiate pulmonary carcinogenesis in animals. We observed differential susceptibility to B[a]P in two human lung adenocarcinoma cell lines, A427 and CL3. DNA adducts were induced by B[a]P treatment in CL3 cells, however, A427 cells were much less responsive to B[a]P treatment. Cytochrome P450 1A1 (CYP1A1) is involved in bioactivation of B[a]P in nonhepatic tissues. Cotreatment with alpha-naphthoflavone, a CYP1A1 inhibitor, abolished DNA adduct formation by B[a]P in CL3 cells. Nevertheless, CYP1A1 inducer beta-naphthoflavone, enhanced DNA adduct formation by B[a]P in both A427 and CL3 cells. Both enzyme activity and mRNA levels of CYP1A1 were highly induced by 1 or 10 microM B[a]P treatment for 24 hr in CL3 cells but not in A427 cells. Protein levels of AhR and aryl hydrocarbon receptor nuclear translocator (Arnt) were similar in A427 and CL3 cells before B[a]P treatment. However, B[a]P induced a retarded band with the [32P]-dioxin responsive element in CL3 cells, but not in A427 cells. This study demonstrated that variation in AhR-mediated CYP1A1 induction contributes to differential susceptibility to B[a]P-DNA adduct formation in human lung cells. Since AhR and/or Arnt function is impaired in A427 cells, this cell line offers a model for investigating the repression mechanisms of CYP1A1 induction by B[a]P in lung cells.     

Genetic restrictions in the development of antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 by nude mice implanted with semiallogeneic thymus glands

Athymic nude mice implanted with F1 thymus glands were used to investigate genetic restrictions regulating T cell-macrophage (M phi) interactions in the development of antibody responses to GAT. Spleen cells from conventional mice developed comparable primary plaque-forming cell (PFC) responses when stimulated by syngeneic and allogeneic GAT-M phi. However, spleen cells from strain A nude mice implanted with (A X B)F1 thymus glands were tolerant of strain B alloantigens and developed GAT-specific PFC responses to strain A GAT-M phi and allogeneic strain C GAT-M phi, but failed to respond to strain B GAT-M phi. The lack of primary GAT-specific PFC responses by spleen cells from (A X B)thy----A nude mice stimulated by strain B GAT-M phi was not due to detectable suppressor mechanisms. However, an allogeneic effect stimulated by H-2- or non-H-2-disparate GAT-pulsed or unpulsed M phi was able to overcome the inability of spleen cells from (A X B)F1 thy----A nude mice to respond to strain B GAT-M phi. Furthermore, the inability to respond to strain B GAT-M phi was overcome by the addition of supernatant fluids from independent cultures of H-2-disparate cells. These results 1) demonstrate that T cells from A nude mice implanted with (A X B)F1 thymus glands did not recognize nominal antigen in the context of B MHC antigens, and 2) suggested that the T cell repertoire was altered in strain A nude mice implanted with (A X B)F1 thymus glands, such that T cells that could recognize GAT in association with strain B MHC antigens were functionally deleted.     

Molecular Basis of Transferrin Polymorphism in Goldfish (Carassius auratus)

Transferrin (TF) polymorphism was investigated in a color variety of goldfish (Carassius auratus), and its molecular basis analyzed. Three TF variants (A1, A2 and B1) were identified from an inbred strain of the goldfish, of which A1 and B1 displayed a large electrophoretic difference on both native and SDS-PAGE gels. The TF cDNAs corresponding to variants A1 and B1 were cloned and sequenced from A1A1, A1B1 and B1B1 individuals, and their deduced amino acid sequences were analyzed. Substantial amino acid variation occurred between variants A1 and B1, with significant differences in peptide length, theoretical molecular weight (Mw) and isoelectric point (pI). No potential glycosylation sites were observed in the two amino acid sequences, which excluded the possibility that carbohydrate difference might cause electrophoretic variation among the TF variants. Further analysis suggested that the distinct electrophoretic mobility of the two variants A1 and B1 by SDS-PAGE resulted from their Mw difference, while the difference by the native PAGE could be explained by their pI variation. Furthermore, genomic DNA fragments containing the transferrin alleles were amplified and subjected to RFLP analysis in A1A1, A1B1 and B1B1 individuals. The data revealed characteristic banding patterns for each TF genotype, and demonstrated that the TF alleles A1 and B1 could be used as a co-dominant marker system. The initial work relating to the goldfish TF variants will benefit the understanding of the evolutionary and functional significance of TF polymorphism in fish.