用膨胀床金属亲和层析从淡菜匀浆液中分离纯化纤维素酶

研究了一种新的膨胀床金属亲和层析技术,即将金属亲和层析结合膨胀床层析,直接从淡菜(Blue mussel)匀浆液中纯化纤维素酶。研究了金属亲和配基种类、pH、离子强度及流速对酶吸附和解吸的影响,确定了酶洗脱条件和介质再生条件。一步可纯化纤维素酶194倍,酶收率达82%。本方法不需要预先去除细胞碎片,而且处理速率比传统层析技术高3～4倍。     

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2)

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.     

沙枣花蜜腺的发育解剖学研究

沙枣的花蜜腺位于花柱基部的筒状花盘上,属花盘蜜腺,其蜜腺位于花盘外方,由分履表皮和产蜜组织组成。分泌表皮具有角质层和变态的气孔器。产蜜组织在发育过程中,其液泡和淀粉粒都随着蜜腺的发育呈现一定的消长规律,最后形成的蜜汁由盘状蜜腺表面的气孔泌出。     

Preclinical Evaluation of Novel Triphenylphosphonium Salts with Broad-Spectrum Activity

Background

Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers.

Methodology/Principal Findings

A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC₅₀ concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation.

Conclusions/Significance

Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling.     

Construction and expression of mouse thymidylate synthase minigenes

Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.     

Oxidative stress-induced leaky sarcoplasmic reticulum underlying acute heart failure in severe burn trauma

Burn trauma causes cardiac dysfunction. However, much of the underlying cellular and molecular mechanisms remain elusive. In the present study, we demonstrate the roles of excessive sarcoplasmic reticulum (SR) Ca(2+) leakage and oxidative stress in burn-associated acute heart failure. In cardiomyocytes from failing rat hearts 12 h after full-thickness cutaneous burn of about 40% of the total body surface area, we found that Ca(2+) transients and contractility were impaired, but the triggering L-type Ca(2+) channel current density was unaltered, giving rise to a significantly reduced gain of excitation-contraction coupling. This deficiency in SR Ca(2+) release was accompanied by a reduction in Ca(2+) content in the SR. Surprisingly, the frequency of spontaneous Ca(2+) sparks was increased by 1.4-fold; Ca(2+) tolerance test (10 mM extracellular Ca(2+)) further showed 2.0- and 1.5-fold more frequent Ca(2+) waves and Ca(2+) sparks, respectively. Myofilament sensitivity to Ca(2+), however, seemed to be unaffected. These results suggest hyperactivity of the ryanodine receptor (RyR) Ca(2+) release channel and a leaky SR in burn. Importantly, pretreatment with antioxidant vitamins C and E seemed to prevent burn-induced RyR hypersensitivity and SR leakage and thereby normalize Ca(2+) transients and contractility. Concomitantly, the in vivo cardiac functions were also more tolerant of traumatic burn. Collectively, our findings suggest that SR leakage due to oxidative stress is likely a major candidate mechanism underlying burn-associated acute heart failure. Antioxidant therapy in burn trauma provides cardioprotection, at least in part, by protecting RyR's from oxidative stress-induced hypersensitivity.     

Quantitative determination of erythromycylamine in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study of dirithromycin

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 degrees C, and the residue was dissolved in 100 microL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm x 2.1 mm I.D., 5 microm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6-9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.     

Genome sequence of Yersinia pestis KIM

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.     

论提高广西桂林漓江上游水源径流量的可能性

论提高广西桂林漓江上游水源径流量的可能性邓世宗唐俊（广西农业大学林学院,南宁５３０００１）（广西林业勘测设计院,南宁５３００１１）ＰｏｏｉｂｉｌｉｔｙｏｆＩｎｃｒｅａｓｉｎｇＷａｔｅｒＤｉｓｃｈａｒｇｅｉｎＵｐｐｅｒＲｅａｃｈｅｓｏｆＬｉｊｉａｎｇＲ...     

Characterization of two monoclonal antibodies, 38F10 and 44D11,against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor(F_0) and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies(mAbs) against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus(HearNPV)(Ha F) were generated. Two kinds of mAbs were obtained according to their different recognition epitopes: one kind of mAbs, as represented by 38F10,recognizes amino acid(aa) 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process.