On-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma with a weak ion exchange monolithic column

A weak ion exchange monolithic column prepared by modifying the GMA-MAA-EDMA (glycidyl methacrylate-methacrylic acid-ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C(18) column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.     

1H NMR investigation on interaction between ibuprofen and lipoproteins

A large number of studies indicate that oxidative modification of plasma lipoproteins, especially low-density lipoprotein (LDL), is a critical factor in initiation and progression of atherosclerosis. We have previously found that ibuprofen (IBP), a potential antioxidant drug to inhibit LDL oxidation, interacted with lipoproteins in intact human plasma. In the present study, we compare the binding affinities of IBP to LDL and HDL (high-density lipoprotein) by (1)H NMR spectroscopy. When IBP is added into the HDL and LDL samples, the - N(+)(CH(3))(3) moieties of phosphatidylcholine (PC) and sphingomyelin (SM) in lipoprotein particles experience the chemical shift up-field drift. Intermolecular cross-peaks observed in NOESY spectra imply that there are direct interactions between ibuprofen and lipoproteins at both hydrophobic and hydrophilic (ionic) regions. These interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group ( - N(+)(CH(3))(3)) and - (CH(2))(n) - group in HDL than that in LDL. This could be explained by either IBP has higher binding affinity to HDL than to LDL, or IBP induces orientation of the phospholipid head group at the surface of the lipoprotein particles.     

Sequence context- and temperature-dependent nucleotide excision repair of a benzo[a]pyrene diol epoxide-guanine DNA adduct catalyzed by thermophilic UvrABC proteins

The influence of DNA base sequence context on the removal of a bulky benzo[a]pyrene diol epoxide-guanine adduct, (+)-trans-B[a]P-N2-dG (G*), by UvrABC nuclease from the thermophilic organism Bacillus caldotenax was investigated. The lesion was flanked by either T or C in otherwise identical complementary 43-mer duplexes (TG*T or CG*C, respectively). It was reported earlier that in the CG*C context, a dominant minor groove adduct structure was observed by NMR methods with all Watson-Crick base pairs intact, and the duplex exhibited a rigid bend. In contrast, in the TG*T context, a highly flexible bend was observed, base pairing at G*, and two 5'-base pairs flanking the adduct were impaired, and multiple solvent-accessible adduct conformations were observed. The TG*T-43-mer duplexes are incised with consistently greater efficiency by UvrABC proteins from B. caldotenax by a factor of 2.3 +/- 0.3. The rates of incisions increase with increasing temperature and are characterized by linear Arrhenius plots with activation energies of 27.0 +/- 1.5 and 23.4 +/- 1.0 kcal/mol for CG*C and TG*T duplexes, respectively. These values reflect the thermophilic characteristics of the UVrABC nuclease complex and the contributions of the different DNA substrates to the overall activation energies. These effects are consistent with base sequence context-dependent differences in structural disorder engendered by a loss of local base stacking interactions and Watson-Crick base pairing in the immediate vicinity of the lesions in the TG*T duplexes. The local weakening of base pairing interactions constitutes a recognition element of the UvrABC nucleotide excision repair apparatus.     

Microarray-based analysis of changes in diversity of microbial genes involved in organic carbon decomposition following land use/cover changes

To increase our understanding of the impact of land use/cover changes on soil microbial decomposition genes involved in organic carbon decomposition, we analyzed soil samples in four sites with different land cover/use histories in a subalpine region of western Sichuan. One site was in a primitive Abies faxoniana forest, the second and the third sites were spruce plantations established in 1960's and 1980's, respectively, and the fourth site was in a cropland dating back to 1960's. The genomic DNA from the microbial community was isolated and hybridized against a functional gene microarray containing 1,961 probes. There were 39, 62, 41, and 28 gene probes with statistically significant positive signals and the gene diversity index (H') values were 3.59, 4.04, 3.70 and 3.16 in primitive forest, spruce plantations established in 1960s and 1980s and cropland, respectively. The results suggested that the number of functional genes and the gene diversity index were correlated with increasing amounts of soil organic carbon, except in the primitive Abies faxoniana forest site. cluster analysis demonstrated that primitive forest soil was clustered more closely to soil from the spruce plantation established in 1960s.     

The nuclear localization signal of mitotic kinesin-like protein Mklp-1: effect on Mklp-1 function during cytokinesis

The mitotic kinesin-like protein (Mklp-1) localizes in the nucleus during interphase due to the presence of nuclear localization signal(s) [NLS(s)] within its sequence. Here, we mapped two NLSs to be 899SRKRRSST906 and 949KRKKP953 in the tail domain of Mklp-1, and showed that ectopic expression of a mutant Mklp-1 without the NLSs leads to cell cycle arrest at cytokinesis, indicating that the NLSs are necessary for Mklp-1 to execute its normal function during cell division. Furthermore, mutation of two serine residues in the first NLS to aspartic acid, which mimics phosphorylation, attenuated its nuclear localization function, suggesting that the function of this NLS might be regulated by phosphorylation.     

Fe binding properties of two soybean (Glycine max L.) LEA4 proteins associated with antioxidant activity

Late embryogenesis abundant (LEA) group 4 (LEA4) proteins play an important role in the water stress tolerance of plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, the protective functions and mechanisms of LEA4 proteins are still not clear. In this study, the metal binding properties of two related soybean LEA4 proteins, GmPM1 and GmPM9, were tested using immobilized metal ion affinity chromatography (IMAC). The metal ions Fe(3+), Ni(2+), Cu(2+) and Zn(2+) were observed to bind these two proteins, while Ca(2+), Mg(2+) or Mn(2+) did not. Results from isothermal titration calorimetry (ITC) indicated that the binding affinity of GmPM1 for Fe(3+) was stronger than that of GmPM9. Hydroxyl radicals generated by the Fe(3+)/H(2)O(2) system were scavenged by both GmPM1 and GmPM9 in the absence or the presence of high ionic conditions (100 mM NaCl), although the scavenging activity of GmPM1 was significantly greater than that of GmPM9. These results suggest that GmPM1 and GmPM9 are metal-binding proteins which may function in reducing oxidative damage induced by abiotic stress in plants.     

Taccasubosides A-D, four new steroidal glycosides from Tacca subflabellata

By analyzing the steroidal content of fresh whole plants of Tacca subflabellata (Taccaceae), we isolated one sapogenin and eight glycosides with four kinds of steroidal skeletons including four new glycosides, named taccasubosides A-D (1-4), together with five known compounds. Among them, compound 1 is the first pentacyclic sterol glycoside with 6-6-6-5-6 fused rings. The structures of 1-4 were elucidated on the basis of extensive spectroscopic analysis, including that of 2D NMR data, and the results of acidic hydrolysis. The cytotoxicity of the selected steroidal glycosides (1-4, 8, and 9) was evaluated in vitro against five human cancer cell lines. Compound 9 showed significant inhibitory activity against all five cell lines.     

Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins

Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.     

Alphaproteobacteria dominate active 2-methyl-4-chlorophenoxyacetic acid herbicide degraders in agricultural soil and drilosphere

2-Methyl-4-chlorophenoxyacetic acid (MCPA) is a widely used phenoxyalkanoic acid herbicide and subject to aerobic microbial degradation. Earthworms stimulate both growth and activity of MCPA-degrading bacteria in soil. Thus, active MCPA degraders in soil and drilosphere (i.e. burrow walls, gut content and cast) were assessed by 16S rRNA stable isotope probing in soil columns under experimental conditions designed to minimize laboratory incubation biases. Agriculturally relevant concentrations of [(13) C]MCPA (20 μg g(dw) (-1)) were degraded in soil within 23 and 27 days in the presence and absence of earthworms respectively. Total 16S rRNA analysis revealed 73 operational taxonomic units indicative of active Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Planctomycetes, Proteobacteria and Verrucomicrobia in soil and drilosphere derived material. Seven operational taxonomic units indicative of Alpha-, Beta-, Gammaproteobacteria and Firmicutes consumed MCPA-[(13) C]. Dominant consumers of MCPA-[(13) C] were Alphaproteobacteria (Sphingomonadaceae and Bradyrhizobiaceae) in soil and drilosphere. Beta- (Comamonadaceae) and Gammaproteobacteria (Xanthomonadaceae) were also important MCPA-[(13) C] consumers in burrow walls only, indicating that earthworms favour betaproteobacterial MCPA degraders. In oxic microcosms with bulk soil, burrow walls and cast, 20 and 300-400 μg g(dw) (-1) [(13) C]MCPA were consumed within 24 h and 20 days respectively. Gut contents did not facilitate the degradation of [(13) C]MCPA. Sphingomonadaceae dominated MCPA-[(13) C] consumers in bulk soil and burrow wall microcosms, while Beta- and Gammaproteobacteria (Burkholderiacea, Comamonadaceae, Oxalobacteraceae and Xanthomonadaceae) dominated MCPA-[(13) C] consumers in microcosms of cast, indicating that the latter taxa are prone to respond to MCPA in cast. The collective data indicated that Alphaproteobacteria are major MCPA degraders in soil and drilosphere.     

Synthesis, characterization, and in vivo biodistribution of 125I-labeled Dex-g-PMAGGCONHTyr

Dextran graft poly (N-methacryloylglycylglycine) copolymer-tyrosine conjugates (dextran-g-PMAGGCONHTyr) were synthesized and characterized. Dynamic light scattering (DLS) results indicated that the graft copolymers are soluble in pH 7.4 PBS and 0.9% saline solutions. The graft copolymers were labeled with (125)I, and the labeling stability in 0.9% saline solution was investigated. Pharmacokinetics studies showed a rapid clearance of (125)I-labeled graft copolymers from the blood pool. Biodistribution images confirmed the preferable liver and spleen accumulation within 1 h after injection and rapid clearance from all the organs over time. The graft copolymer with molecular weight of 9.8 kDa was eliminated from the kidney significantly faster than those with higher molecular weight. The effect of the numbers of -COOH groups on the graft copolymers on the biodistribution was also investigated. It was found that the graft copolymers with the average number of -COOH groups per glucopyranose unit (DS(-COOH)) of 0.57 and 0.18 are mainly distributed in liver and spleen at 1 h after injection, whereas the graft copolymer with DS(-COOH) of 0.07 is mainly accumulated in kidney.