Sesquiterpene lactones of Calea zacatechichi and C. urticifolia

Calea zacatechichi yielded the sesquiterpene lactone zexbrevin and a new analog, several analogs of neurolenin B including calein A, two analogs of budlein A and the flavones acacetin and O-methylacacetin. Calea urticifolia contained additional analogs of neurolenin B as well as a series of epoxidation products. Structures were established by spectrometric techniques. The results are contrasted with previous findings.     

Sesquiterpene lactones from Eremanthus species

The investigation of three Eremanthus species afforded, in addition to known compounds, several new sesquiterpenelactones, a cumambrin B isobutyrate, four lactones related to the eremantholides, two to zexbrevin and one to goyazensolide. Furthermore, a new coniferyl alcohol derivative was isolated. The overall picture of the genus is very uniform. The occurrence of eremanthine and germacranolides with a furanone ring seems to be especially typical.     

Stuessya (Asteraceae: Heliantheae), a new genus from southcentral Mexico

Stuessya, a new genus from the Pacific slopes of southcentral Mexico is described. It is comprised of three species:S. apiculata, previously assigned to the genusViguiera;S. perennans, the generotype; andS. michoacana. The latter two taxa are previously undescribed. The genus is characterized by its urceolate, sclerified (at maturity) involucre. Chromosome counts of2n = 34 are reported forS. perennans. Relationships are problematical, but are reckoned to be somewhere betweenAldama andViguiera.     

Sesquiterpene lactones from Viguiera hypargyrea

The new sesquiterpene lactones 8β-[epoxyangeloyloxy]-14-hydroxytithifolin and 8-[epoxyangeloyl]-14-acetoxy-eupatolide, were isolated from Viguiera hypargyrea. The structures were established by chemical and spectroscopic means. The chemotaxonomic implications of the chemical constituents of Viguiera are briefly discussed.     

Polymyxin B-phosphatidylglycerol interactions. A monolayer (π, ΔV) study

Through a monolayer investigation (π, ΔV), it is shown that the cationic antibiotic polymyxin B (or E) strongly interacts with films of acidic lipids, namely the didodecanoyl- and dihexadecanoylphosphatidylglycerol. The zwitterionic dihexadecanoylphosphatidylcholine was an unsuitable substrate. Interactions occurred at and above a polymyxin B concentration in the subphase of 2.5 · 10^?7 M, bringing about a considerable increase of both π and ΔV. These interactions proceeded in two steps, as revealed by a biphasic change of ΔV with time. They were independent of the film molecular packing (fluid or gel states) and of the initial film pressure.Since it was possible to monitor the relative number of polymyxin B and didodecanoyl- or dihexadecanoylphosphatidylglycerol molecules in the monolayer, it is demonstrated that, at saturation, one polymyxin B molecule is bound to five phosphatidylglycerol molecules, a result which accounts for an exact neutralization of the charges.From competition experiments, it is shown that Na⁺ is ineffective in removing polymyxin B from the interface. Ca²⁺ appeared to be a stronger competitor but no complete antibiotic desorption was observed even at a Ca²⁺ concentration of 100 mM.As a working hypothesis, the antibiotic/lipid ($\text{1}\text{5}$) system was assumed to constitute by itself one molecular species. The mixing of the polymyxin B/didodecanoylphosphatidylglycerol ($\text{1}\text{5}$) system with an excess of lipid molecules in the monolayer was found to be ideal both in terms of π and ΔV. With dihexadecanoylphosphatidylglycerol, a small condensing effect could be detected only at intermediate surface pressures, in a region where the lipid phase transition occurred.The molecular area of polymyxin B interacting with didodecanoylphosphatidylglycerol can be calculated to be 1.23 ± 0.05 nm². It is proposed that the whole antibiotic molecule penetrates the film, the five bound lipid molecules being distributed around the peptide structure, at given positions imposed by the five 2,4-diaminobutyric acid residues.     

RESTRICTION FRAGMENT ANALYSIS OF CHLOROPLAST DNA AND THE SYSTEMATICS OF VIGUIERA AND RELATED GENERA (ASTERACEAE: HELIANTHEAE)

Phylogenetic relationships among 31 species representing variously Helianthus, Helianthopsis, Heliomeris, Simsia, Viguiera, and Tithonia were assessed by chloroplast DNA restriction site mapping. A total of 147 mutations including 92 informative ones was detected using 16 restriction enzymes, with an estimated sequence divergence within the group of 1.4%. Parsimony analysis produced a single most parsimonious tree with a length of 161 steps and a consistency index of 0.91. Statistically significant clades, as assessed by the bootstrap method, correspond to a number of taxa recognized previously, including Helianthus (3 species), Helianthopsis (5 species), and several groups within Viguiera, including sect. Maculatae (4 species), the Baja California group (6 species), sect. Paradosa (2 species) and V. dentata (3 samples). However, species of Viguiera collectively form a highly paraphyletic group relative to species of other genera. Helianthus and Helianthopsis were separated into different clades, supporting their recent segregation. Placement of H. porteri in Helianthus rather than Heliomeris was confirmed; the single sample of the latter genus was most similar to the Baja California group of Viguiera. An expected relationship between Simsia (2 species) and one member of Viguiera ser. Grammatoglossae was confirmed (although not with two other putatively related members of Viguiera) and an unexpected relationship between Simsia and Tithonia was suggested. The presence of Mexican taxa as the more basal groups in the tree points toward a possible Mexican origin for Viguiera and related genera. A molecular clock hypothesis is rejected in many pairwise comparisons involving woody taxa with herbaceous ones, although it could not be rejected in most pairwise comparisons involving taxa of similar habit.     

Germacranolides and diterpenes from Viguiera species

The investigation of six Viguiera species afforded in addition to known compounds two new diterpenes, the 9,11-dehydro derivatives of trachylobanic and stachenic acid, two new heliangolides closely related to viguiestenin, and the acetate of falcarinol. The chemotaxonomic situation is discussed briefly.     

Induction of IL-10-producing regulatory B cells following Toxoplasma gondii infection is important to the cyst formation

Toxoplasma gondii is a protozoan parasite that infects humans and animals via congenital or postnatal routes. During parasite infection, IL-10-producing Bregs are stimulated as part of the parasite-induced host immune responses that favor infection. In this study, we investigated whether T. gondii infection induces immune regulatory cells including IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and whether Breg induction is critical for the development of chronic infection of T. gondii. Furthermore, B cell-deficient (μMT) mice revealed that the IL-10-producing B cells might be associated with the development of chronic T. gondii infection. To better understand the mechanism underlying the accumulation of IL-10-producing B cells upon T. gondii infection, we determined the effect of products released by T. gondii on the induction and differentiation of IL-10-producing B cells during the acute stage of infection using transgenic green fluorescent protein (GFP)-expressing T. gondii strain. We demonstrated that products secreted at the stage of cell lysis by fully replicated tachyzoites induced the differentiation of naive B cells to IL-10-producing Bregs. Our results indicated that the downregulation of the immune response via Bregs during T. gondii infection is related to cyst formation in the host brain and to the establishment of chronic infection.     

Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-¹³C] and [2-¹³C] acetate feeding experiments. ¹³C NMR analyses of the methyl esters of the csypyrone B compounds fed with the ¹³C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus.     

Interleukin-15 expression affects homeostasis and function of B cells through NK cell-derived interferon-γ

Interleukin-15 (IL-15) is a cytokine important for the development, maturation, and function of many cells of the immune system including NK, NKT, γδT, and CD8⁺ T cells. The relationship between IL-15 and B lymphocytes however, is not well characterized and is the focus of our study. Previous in vitro reports have shown that IL-15 increases proliferation of B lymphocytes and increases antibody secretion however, this relationship remains inadequately defined in vivo. The focus of this study was to examine the role of IL-15 in B cell homeostasis and function in vivo using mice that either over express IL-15 (IL-15tg mice) or are deficient in IL-15 (IL-15^−/− mice) production. Here we report significant differences between the B cell populations of IL-15^−/−, C57BL/6, and IL-15tg mice. In fact, increased expression of IL-15 resulted in a significant decrease in the percentage and absolute number of CD19⁺ cells. In vitro B cell co-cultures implicate interferon-gamma (IFN-γ) as the factor responsible for inhibiting B cell proliferation. We also show that IL-15 expression affects B cell function, as B cells from IL-15 transgenic mice produce greater amounts of IgG and IgA than IL-15 knockout mice in vitro. Interestingly, despite significant differences in B cell numbers in these strains, there were no significant differences in total antibody titers in serum and vaginal washes of these mice. Results from our in vivo and in vitro experiments suggest that altered expression of IL-15 affects B cell homeostasis through the induction of NK cell-derived IFN-γ.     

Helicobacter-stimulated IL-10-producing B cells suppress differentiation of lipopolysaccharide/Helicobacter felis-activated stimulatory dendritic cells

Regulatory B cells (Bregs) produce antiinflammatory cytokines and inhibits proinflammatory response. Recently, immunosuppressive roles of Bregs in the effector functions of dendritic cells (DCs) were demonstrated. However, cross talk between Bregs and DCs in Helicobacter infection remains unknown. Here, we showed that direct stimulation of bone marrow-derived DCs (BM-DCs) with Helicobacter felis (H. felis) antigen upregulates their CD86 surface expression and causes the production of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and interleukin-10 (IL-10). Furthermore, prestimulation of DCs with supernatants derived from both Helicobacter-stimulated IL-10^– B (Hf_stim-IL-10^– B) or IL-10⁺ B (Hf_stim-IL-10⁺) cells suppresses the secretion of TNF-α and IL-6, but does not affect the expression of CD86 and secretion of IL-12 by lipopolysaccharide (LPS) or H. felis-activated BM-DCs. Remarkably, soluble factors secreted by Hf_stim-IL-10^– B cells, but not by Hf_stim-IL-10⁺ B cells, suppress the secretion of IL-10 by BM-DCs upon subsequent LPS stimulation. In contrast, prestimulation with BM-DCs with supernatants of Hf_stim-IL-10⁺ B cells before H. felis antigen stimulation induces significantly their IL-10 production. Collectively, our data indicated that prestimulation with soluble factors secreted by Hf_stim-IL-10⁺ B cells, DCs exhibit a tolerogenic phenotype in response to LPS or Helicobacter antigen by secreting high levels of IL-10, but decreased levels of IL-6 and TNF-α.     

Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu³⁵⁹-Lys³⁷³), peptide II (Leu³⁰-Arg⁴⁸), peptide III (Phe¹²⁷-Arg¹⁴⁰), and peptide IV (Arg⁴³⁴-Arg⁴⁴³). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg¹³³, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.     

Inhibitory effects of superoxide dismutase 3 on IgE production in B cells

Immunoglobulin E (IgE) functions as a first-line defense against parasitic infections. However, aberrant production of IgE is known to be associated with various life-threatening allergic diseases. Superoxide dismutase 3 (SOD3) has been found to suppress IgE in various allergic diseases such as allergic conjunctivitis, ovalbumin-induced allergic asthma, and dust mite-induced atopic dermatitis-like skin inflammation. However, the role of SOD3 in the regulation of IgE production in B cells remains elusive. In this study, we investigated the effect of SOD3 on LPS/IL-4 and anti-CD40/IL-4-mediated secretion of IgE in murine B cells. Our data showed that SOD3 can suppress both LPS/IL-4 and antiCD40/IL-7-induced IgE secretion in B cells isolated from both wild-type (SOD3^+/+) and SOD3 knock-out (SOD3^?/?) mice. Interestingly, B cells isolated from SOD3^?/? mice showed higher secretion of IgE, whereas, the use of DETCA, a known inhibitor of SOD3 activity, reversed the inhibitory effect of SOD3 on IgE production. Similarly, SOD3 was found to reduce the proliferation, IgE isotype switch, ROS level, and CCL17 and CCL22 productions in B cells. Furthermore, SOD3 was found to suppress both LPS/IL-4 and anti-CD40/IL-4-mediated activation of downstream signaling such as JAK1/JAK3, STAT6, NF-κB, p38, and JNK in B cells. Taken together, our data showed that SOD3 can be used as an alternative therapy to restrict IgE-mediated allergic diseases.     

Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1–12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC₅₀s of (2.4–52.5?µM), and α-glucosidase with IC₅₀ values of (1.7–72.7?µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC₅₀?=?2.4?µM for 3, 2.8?µM for 7) and α-glucosidase (IC₅₀?=?4.8?µM for 3, 1.7?µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: K_i^app?=?2.4?µM; k₅?=?0.05001?µM^?1?S^?1 and k₆?=?0.02076?µM^?1?S^?1.     

Insect antifeedant secoaromadendrane-type sesquiterpenes from Plagiochila species

The characteristic pungency of the liverworts Plagiochila species P. fruticosa, P. hattoriana, P. ovalifolia and P. yokogurensis is due to a new ent-secoaromadendrane-type sesquiterpene hemiacetal, plagiochiline A, which exhibits very strong antifeedant activity against the African army worm, Spodoptera exempta at 1–10ng/cm². Two new secoaromadendranes, plagiochilide and furanoplagiochilal A, together with the previously known plagiochiline C were isolated from P. yokogurensis. Plagiochilal A, which may be a precursor of plagiochilide and its related hemiacetals, and a bitter principle, plagiochiline B were also isolated from P. hattoriana. P. ovalifolia contained plagiochilines A, B and C. From P. fruticosa, plagiochilide and plagiochilines A, B and C were isolated. The structures of the new secoaromadendrane-type sesquiterpenes were elucidated by extensive ¹H NMR and ¹³CNMR studies.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65^−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65^+/+ cells. The ability of RelA/p65^−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65^−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-κB. Altogether, our findings present a fresh perspective on the role of NF-κB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.     

Immune Correlates of Resistance to Trichinella spiralis Reinfection in Mice

The immune correlate of host resistance induced by reinfection of Trichinella spiralis remains unclear. In this study, we investigated immune correlates between the resistance and serum IgG antibody level, CD23⁺ IgM⁺ B cells, and eosinophil responses induced by T. spiralis reinfection. Mice were primarily infected with 10 or 100 T. spiralis larvae (10 TS, 100 TS), respectively, and after 4 weeks, they were challenge infected with 100 T. spiralis larvae (10–100 TS, 100-100 TS). Upon challenge infections, 10–100 TS mice induced significantly higher levels of T. spiralis-specific total IgG antibody responses in sera and antibody secreting cell responses in spleens compared to 100-100 TS mice, resulting in significantly reduced worm burdens in 10–100 TS mice (60% and 70% reductions for adult and larvae, respectively). Higher levels of eosinophils were found in mice primarily infected with 10 TS compared to those of 100 TS at week 8 upon challenge. CD23⁺ IgM⁺ B cells were found to be increased significantly in mice primarily infected with 10 TS. These results indicate that primary infection of 10 larvae of T. spiralis, rather than 100 larvae, induces significant resistance against reinfection which closely correlated with T. spiralis-specific IgG, eosinophil, and CD23⁺ IgM⁺ B cell responses.