拐芹根化学成分研究Ⅱ

从伞型科当归属植物拐芹(Angelica polymorpha Maxim)的根及根茎中又分得4个结晶性化合物。经物理常数测定、光谱分析,分别鉴定为欧前胡素Ⅰ,异氧化前胡内酯Ⅱ,Pabulenol Ⅲ,Phellopterin Ⅳ。     

Characterization of the basolateral membrane conductance of Necturus urinary bladder

Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that permitted rapid changes in the ion composition of the serosal solution. The transepithelial electrical properties exhibited a marked seasonal variation that could be attributed to variations in the conductance of the shunt pathway, apical membrane selectivity, and basolateral Na+ transport. In contrast, the passive electrical properties of the basolateral membrane remained constant throughout the year. The apparent transference numbers (Ti) of the basolateral membrane for K+ and Cl- were determined from the effect on the basolateral membrane equivalent electromotive force of a sudden increase in the serosal K+ concentration from 2.5 to 50 mM/liter or a decrease in the Cl- concentration from 101 to 10 mM/liter. TK and TCl were 0.71 +/- 0.05 and 0.04 +/- 0.01, respectively. The basolateral K+ conductance could be blocked by Ba2+ (0.5 mM), Cs+ (10 mM), or Rb+ (10 mM), but was unaffected by 3,4-diaminopyridine (100 microM), decamethonium (100 microM), or tetraethylammonium (10 mM). We conclude that a highly selective K+ conductance dominates the electrical properties of the basolateral membrane and that this conductance is different from those found in nerve and muscle membranes.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs — these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science — and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.     

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics±antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3±1.7% TI versus 10.2±4.1% TI, n=19), but not of non-smokers (8.6±2.8% TI versus 8.3±2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4±2.9% TI versus 18.9±13.1% TI, n=15) but not in smokers (15.5±10.7% TI versus 20.4±14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread±antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.     

Synthesis and SAR of 1,3-disubstituted cyclohexylmethyl urea and amide derivatives as non-peptidic motilin receptor antagonists

A series of 1,3-disubstituted cyclohexylmethyl urea and amide derivatives were synthesized as motilin receptor antagonists. Starting from known motilin antagonists, 1a and 1b, the cyclopentene scaffold was replaced and the four recognition elements optimized to arrive at a potent novel series.     

Optimization of the antibody C(H)3 domain by residue frequency analysis of IgG sequences

In an attempt to enhance the overall assembly, yield and half-life of recombinant antibody proteins, we have cloned and expressed several IgG1 C(H)3 domains and examined their folding/refolding characteristics. We utilized a cytoplasmic bacterial expression system with a thioredoxin reductase knock-out strain of BL21(DE3) to produce bovine, murine and human C(H)3. Under identical conditions, expression of bovine C(H)3 resulted consistently in the highest yields of properly folded/oxidized protein. Circular dichroism and fluorescence experiments demonstrate that oxidized bovine and murine C(H)3 have surprisingly similar structures and stabilities, considering the marginal sequence conservation between the two molecules. Residue frequency analysis using a limited data set of 36 unique Fc sequences originating from 19 different mammalian species targeted five specific sites for optimization within bovine C(H)3. Combination of three of these mutants increased the thermal stability of the molecule to 86 degrees C. Comparison of this approach to similar studies using larger sequence databases and/or different selection criteria suggests sequence database design can increase the success rate for identifying residue sites worth optimizing. This optimized C(H)3 domain can be used as a particularly stable platform for functional design and can be grafted into full-length antibody sequences to enhance their thermodynamic parameters and shelf-life.     

Discovery of a dihydropyrimidine series of molecules that selectively mimic the biological actions of calcitonin

The use of a multiplex mimetic assay led us to identify 1,4-dihydropyrimidines with potent and selective calcitonin receptor mimetic activity. Subsequent modification of the dihydropyrimidine scaffold led to a series of molecules that were efficacious in a neonatal mouse calvaria in vitro model. Dihydropyrimidine 5h, in particular, was identified as a calcitonin mimetic (EC(50)=6 microM), active in-vivo in the Weanling rat model when administered subcutaneously.