A Novel Eliminase from a Marine Bacterium That Degrades Hyaluronan and Chondroitin Sulfate

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ^4,5HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.     

Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase

Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973–25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans.     

Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30–50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.     

A Liquid Chromatography-Mass Spectrometry-based Approach to Characterize the Substrate Specificity of Mammalian Heparanase

Extracellular heparanase activity releases growth factors and angiogenic factors from heparan sulfate (HS) storage sites and alters the integrity of the extracellular matrix. These activities lead to a loss of normal cell matrix adherent junctions and correlate with invasive cellular phenotypes. Elevated expression of heparanase is associated with several human cancers and with vascular remodeling. Heparanase cleaves only a limited fraction of glucuronidic linkages in HS. There have been few investigations of the functional consequences of heparanase activity, largely due to the heterogeneity and complexity of HS. Here, we report a liquid chromatography-mass spectrometry (LC-MS)-based approach to profile the terminal structures created by heparanase digestion and reconstruct the heparanase cleavage sites from the products. Using this method, we demonstrate that heparanase cleaves at the non-reducing side of highly sulfated HS domains, exposing cryptic growth factor binding sites. This cleavage pattern is observed in HS from several tissue sources, regardless of overall sulfation degree, indicating a common recognition pattern. We further demonstrate that heparanase cleavage of HS chains leads to increased ability to support FGF2-dependent cell proliferation. These results suggest a new mechanism to explain how heparanase might potentiate the uncontrolled cell proliferation associated with cancer through its ability to activate nascent growth factor-promoting domains within HS.     

Partitioning of 14C-Photosynthate of Leaves in Roots,Rhizome, and in Essential Oil and Curcumin in Turmeric (Curcuma longa L.)

Incorporation of photosynthetically fixed ¹⁴C was studied at different time intervals of 12, 24, and 36 h in various plant parts—leaf 1 to 4 from apex, roots, and rhizome—into primary metabolites—sugars, amino acids, and organic acids, and secondary metabolites—essential oil and curcumin—in turmeric. The youngest leaves were most active in fixing ¹⁴C at 24 h. Fixation capacity into primary metabolites decreased with leaf position and time. The primary metabolite levels in leaves were maximal in sugars and organic acids and lowest in amino acids. Roots as well as rhizome received maximum photoassimilate from leaves at 24 h; this declined with time. The maximum metabolite concentrations in the roots and rhizome were high in sugars and organic acids and least in amino acids. ¹⁴C incorporation into oil in leaf and into curcumin in rhizome was maximal at 24 h and declined with time. These studies highlight importance of time-dependent translocation of ¹⁴C-primary metabolites from leaves to roots and rhizome and their subsequent biosynthesis into secondary metabolite, curcumin, in rhizome. This might be one of factors regulating the secondary metabolite accumulation and rhizome development.     

Immunohistochemical Evaluation of Global DNA Methylation: Comparison with in Vitro Radiolabeled Methyl Incorporation Assay

The in vitro radiolabeled methyl incorporation assay, a commonly used technique to evaluate global methylation of DNA, has some disadvantages and limitations. The purpose of the present study was to compare the results of global DNA methylation evaluated by radiolabeled methyl incorporation (CPM/μg of DNA) with immunohistochemical staining of the same tissue sections with a monoclonal antibody developed against 5-methylcytosine (5-mc). We used archival specimens of squamous cell cancer (SCC) of the human lung with a matched uninvolved specimen (n = 18 pairs) and 18 lung specimens from subjects without lung cancer (noncancer specimens) to make this comparison. The immunostaining for 5-mc was reported as a percentage of cells positive for staining as well as a weighted average of the intensity score. The results suggested that both radiolabeled methyl incorporation assay and immunostaining for 5-mc can be used to demonstrate hypomethylation of DNA in SCC tissues compared to matched uninvolved tissues. An advantage of immunostaining, however, is its ability to demonstrate hypomethylation of SCC compared to adjacent bronchial mucosa on the same archival specimen, obviating the need to use sections from both SCC and matched uninvolved tissues. Only by using the immunostaining technique were we able to document a statistically significant difference in DNA methylation between SCC and noncancer tissues. We conclude that the immunostaining technique has advantages over the radiolabeled methyl incorporation assay and may be best suited for evaluation of global DNA methylation when the methylation status of cancer cannot be normalized by methyl incorporation of normal tissues or when the number of samples available for evaluation is small.     

汉阳陵文物表面硫酸盐形成原因微生物学证据

用仅含硫源的无机盐培养基对采自汉阳陵13号和15号遗址坑文物表面的土样进行细菌富集筛选实验,并对分离的菌株进行生理生化特性研究、16SrDNA序列分析及硫代谢能力测试。分离得到5株细菌:TD1、TD2、TD3、TD15及PL13。其中TD1属于Alcaligenes,TD3属于Bacillus;硫代谢能力测试结果显示TD2、TD15和PL13代谢产生硫酸盐的速率远远小于TD3和TD1,TD3氧化硫源生成硫酸盐的速率是TD1的1.4倍。实验证实这些菌株均能氧化S、S2O32-和S2-生成硫酸盐从而获得能量,这表明微生物氧化土壤中硫化物是汉阳陵文物表面硫酸盐增加的原因,从而为汉阳陵文物表面硫酸盐的形成原因提供了微生物学依据。     

Topoisomerase II poisoning by indazole and imidazole complexes of ruthenium

Trans-imidazolium (bis imidazole) tetrachloro ruthenate (RuIm) and trans-indazolium (bis indazole) tetrachloro ruthenate (RuInd) are ruthenium coordination complexes, which were first synthesized and exploited for their anticancer activity. These molecules constitute two of the few most effective anticancer ruthenium compounds. The clinical use of these compounds however was hindered due to toxic side effects on the human body. Our present study on topoisomerase II poisoning by these compounds shows that they effectively poison the activity of topoisomerase II by forming a ternary cleavage complex of DNA, drug and topoisomerase II. The thymidine incorporation assays show that the inhibition of cancer cell proliferation correlates with topoisomerase II poisoning. The present study on topoisomerase II poisoning by these two compounds opens a new avenue for renewing further research on these compounds. This is because they could be effective lead candidates for the development of more potent and less toxic ruthenium containing topoisomerase II poisons. Specificity of action on this molecular target may reduce the toxic effects of these ruthenium-containing molecules and thus improve their therapeutic index.     

Sulfate reduction in methanogenic bioreactors

Abstract: In the anaerobic treatment of sulfate-containing wastewater, sulfate reduction interferes with methanogenesis. Both mutualistic and competitive interactions between sulfate-reducing bacteria and methanogenic bacteria have been observed. Sulfate reducers will compete with methanogens for the common substrates hydrogen, formate and acetate. In general, sulfate reducers have better growth kinetic properties than methanogens, but additional factors which may be of importance in the competition are adherence properties, mixed substrate utilization, affinity for sulfate of sulfate reducers, relative numbers of bacteria, and reactor conditions such as pH, temperature and sulfide concentration. Sulfate reducers also compete with syntrophic methanogenic consortia involved in the degradation of substrates like propionate and butyrate. In the absence of sulfate these methanogenic consortia are very important, but in the presence of sulfate they are thought to be easily outcompeted by sulfate reducers. However, at relatively low sulfate concentrations, syntrophic degradation of propionate and butyrate coupled to HZ removal via sulfate reduction rather than via methanogenesis may become important. A remarkable feature of some sulfate reducers is their ability to grow fermentatively or to grow in syntrophic association with methanogens in the absence of sulfate.