技术资料

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1),6-carbamoyl-7-desacetylforskolins (series 2),and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1,IC50 = 43-1600 nM,EC50 = 0.5-9.6 microM; series 2,IC50 = 65-680 nM,EC50 = 0.63-6.5 microM; series 3,IC50 = 21-271 nM,EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however,some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position,although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however,the effect was more pronounced with binding. For both binding and activation,small,linear,lipophilic substituents (propyl,allyl,isopropyl) are well tolerated at the 7-position but less so in the 6-position,even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents. View Publication

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Reaction with peroxynitrite at pH 7.4 and 37 degrees C was found to increase the 8-oxodeoxyguanosine levels in calf thymus DNA 35- 38-fold. This oxidation of deoxyguanosine,as well as the peroxynitrite-mediated nitration of tyrosine to 3-nitrotyrosine,was significantly inhibited by ascorbic acid,glutathione and (-)-epigallocatechin gallate,a polyphenolic antioxidant present in tea. For 50% inhibition of the oxidation of deoxyguanosine to 8-oxodeoxyguanosine,1.1,7.6 or 0.25 mM ascorbate,glutathione or (-)-epigallocatechin gallate,respectively,was required. For 50% inhibition of tyrosine nitration,the respective concentrations were 1.4,4.6 or 0.11 mM. Thus,(-)-epigallocatechin gallate is a significantly better inhibitor of both reactions than either ascorbate or glutathione. Reaction of (-)-epigallocatechin gallate with peroxynitrite alone resulted in the formation of a number of products. Ultraviolet spectra of two of these suggest that the tea polyphenol and/or its oxidation products are nitrated by peroxynitrite. View Publication

1. The effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of complement 5a (C5a)- and platelet-activating factor (PAF)-stimulated human eosinophils obtained from normal and atopic donors were investigated. 2. Eosinophils were purified from peripheral venous blood of normal and atopic subjects by an immunomagnetic procedure to a purity textgreater 99%. Eosinophils were stimulated with PAF (0.1 microM) or C5a 0.1 microM for 15 min and LTC4 was measured by radioimmunoassay (RIA). Eosinophil chemotaxis in response to PAF and C5a was assessed with 48-well microchambers (Boyden). 3. Under these conditions substantial amounts of LTC4 (about 300-1000 pg per 10(6) cells) were only detectable in the presence of indomethacin (0.1-10 microM). To explain this finding it was hypothesized that indomethacin reversed the inhibition of LTC4 synthesis by endogenously synthesized prostaglandins,in particular prostaglandin E2 (PGE2). In fact,eosinophils release 23 pg PGE2 per 10(6) cells following PAF stimulation; this PGE2 synthesis was completely inhibited by indomethacin and readdition of PGE2 inhibited eosinophil LTC4 synthesis (IC50 = 3 nM). The following experiments were performed in the presence of 10 microM indomethacin. 4. Theophylline (IC50 approximately 50 microM) and rolipram (IC50 approximately 0.03-0.2 microM) suppressed PAF- and C5a-stimulated LTC4 synthesis. This PDE inhibitor-induced suppression of LTC4 generation is mediated by activation of protein kinase A,since it was reversed by the protein kinase A inhibitor Rp-8-Br-cyclic AMPS. In addition,exogenous arachidonic acid concentration-dependently (0.3 microM-3 microM) reversed the inhibition of LTC4 synthesis by the PDE inhibitors,indicating that theophylline and rolipram suppress the mobilization of arachidonic acid. The beta 2-adrenoceptor agonist salbutamol inhibited eosinophil LTC4 synthesis (IC50 = 0.08 microM). The combination of salbutamol with theophylline (10 microM) or rolipram (3 nM) appeared to be additive. 5. Theophylline (IC50 approximately 40 microM),rolipram (IC50 approximately 0.02 microM [C5a],approximately 0.6 microM [PAF]) and PGE2 (IC50 approximately 3 nM) inhibited C5a- and PAF-stimulated eosinophil chemotaxis. The combination of PGE2 with theophylline resulted in an additive effect. 6. Both C5a- and PAF-stimulated eosinophil chemotaxis and LTC4 generation were significantly elevated in eosinophils from atopic individuals compared to normal subjects. However,eosinophils from normal and atopic individuals were not different with respect to their total cyclic AMP-PDE and PDE4 isoenzyme activities as well as the potencies of theophylline and rolipram to suppress LTC4 generation and chemotaxis. View Publication

The inhibitory and bactericidal activities of anacardic acid and totarol,alone and in combination with methicillin,were investigated against methicillin-resistant Staphylococcus aureus (MRSA). The growth of two MRSA strains was inhibited by 6 x 25 microg ml-1 of anacardic acid and 0 x 78 microg ml-1 of totarol. The time-kill curve study showed that these two compounds were bactericidal against MRSA. Anacardic acid killed MRSA cells more rapidly than totarol,and no viable cells were detected after being exposed to 6 x 25 microg ml-1 of anacardic acid for 6 h. Anacardic acid showed bactericidal activity against MRSA at any stage of growth,and also even when cell division was inhibited by chloramphenicol. In the combination studies,the minimal inhibitory concentration (MIC) of methicillin was lowered from 800 to 1 x 56 microg ml-1 for MRSA ATCC 33591,and from 800 to 6 x 25 microg ml-1 for MRSA ATCC 33592,by combining with 1/2 x MIC of anacardic acid. The time-kill curves demonstrated synergistic bactericidal activities for these combinations. View Publication

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes,the co-stimulation of CD4+ T cells,and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells,but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together,these data implicate HSA as a potent negative regulator of B cell development and activation. View Publication

MUC-1 mucin is considered to be aberrantly glycosylated in breast,ovary,and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation,we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1,MTSV1-7,and HB-2) derived from cells cultured from human milk,with three breast cancer cell lines (MCF-7,BT-20,and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1,and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern,with NeuAcalpha2-3Galbeta1-3GalNAc-ol,Galbeta1-3GalNAc-ol,and GalNAc-ol predominating; in contrast,MUC-1 from MTSV1-7 had more complex structures,including a number of disialo,core 2 species,i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler,and fewer,carbohydrate chains than MUC-1 from normal breast epithelial cells,and that these differences,combined or separately,explain the differential tumor specificity of some MUC-1 antibodies and T cells. View Publication

We have identified three non-cross-reacting anti-human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long-term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC-initiating cells (LTC-IC),and the number of colony-forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34+ CD38- normal marrow cells were placed on pre-established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained,and dependent on the presence of a preformed feeder layer. Interestingly,the same anti-CD44 antibodies had no effect on the maintenance of leukaemic (Ph+) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph+) counterparts. View Publication

Adult-derived hippocampal progenitors generate neurons,astrocytes,and oligodendrocytes in vitro and following grafting into the adult brain. Although these progenitors have a considerable capacity for in vitro self renewal,it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking,we have followed individual cells through the process of proliferative expansion,commitment,and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid,elevated cyclic AMP,or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain. View Publication

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases,each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI),respectively,but are insensitive to 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis. View Publication

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene,a codon humanized,red-shifted variant of the green fluorescent protein (GFP) gene,which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6,up to 16% of the cells expressed EGFP,as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers. View Publication

Mitomycin C,an important antitumor drug and antibiotic,has an extraordinary ability to crosslink DNA with high efficiency and absolute specificity for the sequence CpG. Recent results have shown how mitomycin C crosslinks DNA,and why the sequence specificity is so complete. This new understanding may allow the design of agents that mimic mitomycin C's economy of structure and can crosslink other sequences. View Publication