Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts

AIMS: Use of an electronic nose (zNose(TM)) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media. METHODS AND RESULTS: Cultures of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and yeasts (two Candida albicans strains) were grown on agar and in broth media and incubated for 24 h at 37 degrees C. Headspace samples from microbial cultures were analysed by the zNose(TM), a fast gas chromatography-surface acoustic wave detector. Olfactory images of volatile production patterns were observed to be different for the various species tested after 24 h. Moreover, some strains (two K. pneumoniae, two C. albicans) did not show changes in volatile production patterns within our species. CONCLUSIONS: Our experiments demonstrate that the electronic nose system can recognize volatile production patterns of pathogens at species level. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results, although preliminary, promise exciting challenges for microbial diagnostics.     

麦角碱生物合成途径中酶学及相关基因研究进展

简要介绍了麦角碱(ergot alkaloids)的化学、药理学及生物合成方面的相关知识．综述了近年来麦角碱生物合成途径中酶学和相关基因方面的研究进展以及它对麦角碱生产的影响,探讨了麦角碱生物合成途径方面的研究方向和发展前景。     

土地整理过程中碳量损失与生态补偿优化设计

土地整理改变了项目区土地利用结构,打破了生态系统碳循环平衡。在土地整理实施的过程中以及实施以后一段时期,项目区生态系统碳蓄积量必然损失．从而使项目区农业生态系统由碳汇变成了碳源。以湖北省荆门市东宝区崔院村土地整理项目区为例,拟通过对生态系统碳蓄积量的估算,并采用线性规划的方法求算土地整理规划设计的最佳土地利用结构,从而实现对土地整理所引起的生态系统碳量损失进行补偿的目标。结果表明,为了实现土地整理的耕地面积最大化目标,通过采用线性规划的方法所得到的土地利用结构优化方案能够很好地满足规划设计所涉及的各类用地的面积以及生态系统的碳量平衡等约束条件。耕地面积增加量占总土地面积的11．94％实现丁最大化目标．同时满足了道路系统和水利灌溉系统以及居民点等用地的需求,而人工林地的面积必须增加3.90hm^2,也就实现土地整理前后生态系统碳量的平衡。     

Molecular modeling of the non-covalent binding of the dietary tomato carotenoids lycopene and lycophyll, and selected oxidative metabolites with 5-lipoxygenase

Numerous studies on human prostate cancer cell lines indicate a role for arachidonic acid (AA) and its oxidative metabolites in prostate cancer proliferation. The metabolism of AA by either the cyclooxygenase (COX) or the lipoxygenase (LOX) pathways generates eicosanoids involved in tumor promotion, progression, and metastasis. In particular, products of the 5-LOX pathway (including 5-HETE and 5-oxo-EET) have been implicated as potential 'survival factors' that may confer escape after androgen withdrawal therapy through fatty-acid (i.e., AA) drive. Potent natural dietary antioxidant compounds such as lycopene and lycophyll, with tissue tropism for human prostate, have been shown to be effective in ameliorating generalized oxidative stress at the DNA level. Suppressing the 5-LOX axis pharmacologically is also a promising avenue for intervention in human patients. The recently recognized direct interaction of the astaxanthin-based soft-drug Cardax to human 5-LOX with molecular modeling, and the downregulation of both 5-HETE and 5-oxo-EET in vivo in a murine peritonitis model, suggest that other important dietary carotenoids may share this enzyme regulatory feature. In the current study, the acyclic tomato carotene lycopene (in all-trans and 5-cis isomeric configurations) and its natural dihydroxy analog lycophyll (also present in tomato fruit) were subjected to molecular modeling calculations in order to investigate their predicted binding interaction(s) with human 5-LOX. Two bioactive oxidative metabolites of lycopene (4-methyl-8-oxo-2,4,6-nonatrienal and 2,7,11-trimethyl-tetradecahexaene-1,14-dial) were also investigated. A homology model of 5-LOX was constructed using 8-LOX and 15-LOX structures as templates. The model was validated by calculating the binding energy of Cardax to 5-LOX, which was demonstrated to be in good agreement with the published experimental data. Blind docking calculations were carried out in order to explore the possible binding sites of the carotenoids on 5-LOX, followed by focused docking to more accurately calculate the predicted energy of binding. Lycopene and lycophyll were predicted to bind with high affinity in the superficial cleft at the interface of the beta-barrel and the catalytic domain of 5-LOX (the 'cleavage site'). Carotenoid binding at this cleavage site provides the structural rationale by which polyenic compounds could modify the 5-LOX enzymatic function via an allosteric mechanism, or by radical scavenging in proximity to the active center. In addition, the two bioactive metabolites of lycopene were predicted to bind to the catalytic site with high affinity--therefore suggesting potential direct competitive inhibition of 5-LOX activity that should be shared by both lycopene and lycophyll after in vivo supplementation, particularly in the case of the dial metabolite.     

Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains

Proteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration.     

Survival of Environmental Mycobacteria in Acanthamoeba polyphaga

Free-living amoebae in water are hosts to many bacterial species living in such an environment. Such an association enables bacteria to select virulence factors and survive in adverse conditions. Waterborne mycobacteria (WBM) are important sources of community- and hospital-acquired outbreaks of nontuberculosis mycobacterial infections. However, the interactions between WBM and free-living amoebae in water have been demonstrated for only few Mycobacterium spp. We investigated the ability of a number (n = 26) of Mycobacterium spp. to survive in the trophozoites and cysts of Acanthamoeba polyphaga. All the species tested entered the trophozoites of A. polyphaga and survived at this location over a period of 5 days. Moreover, all Mycobacterium spp. survived inside cysts for a period of 15 days. Intracellular Mycobacterium spp. within amoeba cysts survived when exposed to free chlorine (15 mg/liter) for 24 h. These data document the interactions between free-living amoebae and the majority of waterborne Mycobacterium spp. Further studies are required to examine the effects of various germicidal agents on the survival of WBM in an aquatic environment.     

Role of the RANKL/RANK system in the induction of interleukin-8 (IL-8) in B chronic lymphocytic leukemia (B-CLL) cells

B chronic lymphocytic leukemia (B-CLL) cells express several members of the tumor necrosis factor (TNF) family, such as CD40L, CD30L, and TRAIL. By using the cDNA microarray technology, B-CLL samples were found to overexpress receptor activator of nuclear factor kB (NF-kB) ligand (RANKL), as compared to normal CD19(+) B cells. These findings were validated at the protein level by Western blot and flow cytometry analyses. Moreover, unlike primary normal B cells, leukemic B-CLL cells showed surface expression of RANK, the cognate transmembrane receptor of RANKL. When added in vitro to B-CLL cultures, either alone or in association with chlorambucil or fludarabine, recombinant RANKL did not significantly modulate cell viability, and it minimally affected the IL-8 expression/release. On the other hand, treatment with RANK-Fc chimera potently upregulated the release of IL-8 in the B-CLL culture supernatants, suggesting involvement of reverse signaling through transmembrane RANKL in IL-8 induction. In turn, exposure of B-CLL cells to recombinant IL-8 significantly decreased spontaneous apoptosis as well as chlorambucil- and fludarabine-mediated cytoxicity in B-CLL cells. Since IL-8 has been implicated in progression of B-CLL disease, our findings suggest that, by upregulating IL-8, the RANKL/RANK system may contribute to the pathogenesis of B-CLL.     

Interaction of TPPP/p25 protein with glyceraldehyde-3-phosphate dehydrogenase and their co-localization in Lewy bodies

TPPP/p25, a flexible unstructured protein, binds to tubulin and induces aberrant microtubule assemblies. We identified hereby glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a new interacting partner of TPPP/p25. The immunoprecipitation and affinity chromatographic experiments with bovine brain cell-free extract revealed that the interaction was salt and NAD(+) sensitive while ELISA showed resistant and firm association of the two isolated proteins. In transfected HeLa cells at low expression level of EGFP-TPPP/p25, while the green fusion protein aligned at the microtubular network, GAPDH distributed uniformly in the cytosol. However, at high expression level, GAPDH co-localized with TPPP/p25 in the aggresome-like aggregate. Immunohistochemistry showed enrichment of TPPP/p25 and GAPDH within the alpha-synuclein positive Lewy body.     

Role of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulation

A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing>or=1 microM Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3',4'-dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 microM, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50=2.44 microM). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role.