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61.
To obtain insight into the functional properties of Treponema denticola cystalysin, we have analyzed the pH- and ligand-induced spectral transitions, the pH dependence of the kinetic parameters, and the substrate specificity of the purified enzyme. The absorption spectrum of cystalysin has maxima at 418 and 320 nm. The 320 nm band increases at high pH, while the 418 nm band decreases; the apparent pK(spec) of this spectral transition is about 8.4. Cystalysin emitted fluorescence at 367 and 504 nm upon excitation at 320 and 418 nm, respectively. The pH profile for the 367 nm emission intensity increases above a single pK of approximately 8.4. On this basis, the 418 and 320 nm absorbances have been attributed to the ketoenamine and substituted aldamine, respectively. The pH dependence of both log k(cat) and log k(cat)/K(m) for alpha,beta-elimination reaction indicates that a single ionizing group with a pK value of approximately 6.6 must be unprotonated to achieve maximum velocity. This implies that cystalysin is more catalytically competent in alkaline solution where a remarkable portion of its coenzyme exists as inactive aldamine structure. Binding of substrates or substrate analogues to the enzyme over the pH range 6-9.5 converts both the 418 and 320 nm bands into an absorbing band at 429 nm, assigned to the external aldimine in the ketoenamine form. All these data suggest that the equilibrium from the inactive aldamine form of the coenzyme shifts to the active ketoenamine form on substrate binding. In addition, reinvestigation of the substrate spectrum of alpha,beta-elimination indicates that cystalysin is a cyst(e)ine C-S lyase rather than a cysteine desulfhydrase as claimed previously. 相似文献
62.
Clausen VA Bae W Throup J Burnham MK Rosenberg M Wallis NG 《Journal of molecular microbiology and biotechnology》2003,5(4):252-260
The YYCFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (YYCF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K(m )(ATP) of S. aureus YycG autophosphorylation was 130 microM and S. pneumoniae was 3.0 microM. Each had similar K(cat )values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets. 相似文献
63.
Kinarsky L Suryanarayanan G Prakash O Paulsen H Clausen H Hanisch FG Hollingsworth MA Sherman S 《Glycobiology》2003,13(12):929-939
The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases. 相似文献
64.
Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis 总被引:1,自引:0,他引:1
Group B and C Neisseria meningitidis are the major cause of meningococcal
disease in the United States and in Europe. N . meningitidis
lipooligosaccharide (LOS), a major surface antigen, can be divided into 12
immunotypes of which L1 through L8 were found among Group B and C
organisms. Groups B and C but not Group A may sialylate their LOSs with
N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they
synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as
probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4,
L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis
leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc
sequence, but not to Sambucus nigra agglutinin, which binds
NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot
analyses revealed that these six LOSs contained only the
NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS
components, which have a common terminal lacto-N-neotetraose (LNnT,
Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown
by previous studies. The LOS-lectin binding was abolished when the LOSs
were treated with Newcastle disease viral neuraminidase which cleaves
alpha2-->3 linked sialic acid. Methylation analysis of a representative
LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs
structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide)
and sialylparagloboside and some glycoproteins in having LNnT and
N-acetyllactosamine sequences, respectively, with or without alpha2-->3
linked NeuNAc. The molecular mimicry of the LOSs may play a role in the
pathogenesis of N.meningitidis by assisting the organism to evade host
immune defenses in man.
相似文献
65.
66.
Most preservative-treated wood produced and consumed in the United States is treated with toxic inorganic compounds containing
copper, chromium, and arsenic. Because chromated copper arsenate (CCA) is fixed to the wood, CCA-treated wood has not been
considered toxic or hazardous and it is currently disposed of in approved landfills. Growing public concern about environmental
contamination from treated wood combined with the removal of greater quantities of CCA-treated wood from service have presented
a disposal challenge for this fiber source. In this study, CCA-treated wood was processed by acid extraction, steam explosion,
and bacterial fermentation and evaluated for removal of copper, chromium, and arsenic. Copper was the easiest to remove by
these treatments and chromium the most resistant to removal. Exposing CCA-treated wood to steady-state bacterial growth by
continuous culture with Bacillus licheniformis CC01 did not enhance removal of CCA components compared to standard mixed culture when acid extraction preceded bacterial
fermentation. Nor did steam explosion, alone or in conjunction with acid extraction and bacterial fermentation, enhance removal
of CCA components; the chromium and arsenic components resisted removal. Grinding CCA-treated wood chips into 20-mesh sawdust
provided greater access to and removal of CCA components by all processes. However, grinding the chips was unnecessary if
they were treated with acid prior to bacterial fermentation. Extraction with oxalic acid as a precursor to bacterial fermentation
with B. licheniformis CC01 removed 90% copper (CuO), 80% chromium (CrO3), and 100% arsenic (As2O5) from treated chips. The combination of acid extraction and bacterial fermentation removed 80–100% of these metals from CCA-treated
wood.
Received 15 December 1997/ Accepted in revised form 08 March 1998 相似文献
67.
Jens Clausen Dr. 《Biotechnology journal》2008,3(12):1493-1501
Modern brain technology is a highly dynamic and innovative field of research with great potential for medical applications. Recent advances in recording neural signals from the brain by brain-machine interfacing presage new therapeutic options for paralyzed people by means of neural motor prostheses. This paper examines foreseeable ethical questions related to the research on brainmachine interfaces and their possible future applications. It identifies four major topics that need to be considered: first, the questions of personality and its possible alterations; second, responsibility and its possible constraints; third, therapeutic applications and their possible exceedance; and fourth, questions of research ethics that arise when progressing from animal experimentation to application to human subjects. This paper, in identifying and addressing the ethical questions raised by brain-machine interfaces, presents concerns that need to be considered if possible prosthetics based on modern brain technology are to be used cautiously and responsibly. 相似文献
68.
69.
Richard Beatson Gjertrud Maurstad Gianfranco Picco Appitha Arulappu Julia Coleman Hans H. Wandell Henrik Clausen Ulla Mandel Joyce Taylor-Papadimitriou Marit Sletmoen Joy M. Burchell 《PloS one》2015,10(5)
Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar deadadhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours. 相似文献
70.
Lauge Peter Westergaard Clausen Ulrich Gosewinkel Karlson Stefan Trapp 《International journal of phytoremediation》2015,17(4):369-376
The willow tree (Salix viminalis) toxicity test and a cress seed germination test (Lepidium sativum) were used to determine uptake of F and phytotoxicity of NaF. Concentrations in hydroponic solutions were 0–1000 mg F/L and 0–400 mg F/L in the preliminary and definitive test. A third test was done with soils collected from a fluoride-contaminated site at Fredericia, Denmark. The EC10, EC20 and EC50-values for inhibition of transpiration were determined to 38.0, 59.6 and 128.7 mg F/L, respectively. The toxicity test with soil showed strong inhibition for the sample with the highest fluoride concentration (405 mg free F per kg soil, 75 mg F per L soil solution). The seed germination and root elongation test with cress gave EC10, EC20 and EC50-values of 61.4, 105.0 and 262.8 mg F/L, respectively. At low external concentrations, fluoride was taken up more slowly than water and at high external concentrations at the same velocity. This indicates that an efflux pump becomes overloaded at concentrations above 210 mg F/L. Uptake kinetics were simulated with a non-linear mathematical model, and the Michaelis-Menten parameters were determined to half-saturation constant KM near 2 g F/L and maximum enzymatic removal rate vmax at 9 g/(kg d). 相似文献