The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses

Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kDa protein from P. furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6-A resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea).     

PIV-validated numerical modeling of pulsatile flows in distal coronary end-to-side anastomoses

This study employed particle image velocimetry (PIV) to validate a numerical model in a complementary approach to quantify hemodynamic factors in distal coronary anastomoses and to gain more insights on their relationship with anastomotic geometry. Instantaneous flow fields and wall shear stresses (WSS) were obtained from PIV measurement in a modified life-size silastic anastomosis model adapted from a conventional geometry by incorporating a smooth graft-artery transition. The results were compared with those predicted by a concurrent numerical model. The numerical method was then used to calculate cycle-averaged WSS (WSS(cyc)) and spatial wall shear stress gradient (SWSSG), two critical hemodynamic factors in the pathogenesis of intimal thickening (IT), to compare the conventional and modified geometries. Excellent qualitative agreement and satisfactory quantitative agreement with averaged normalized error in WSS between 0.8% and 8.9% were achieved between the PIV experiment and numerical model. Compared to the conventional geometry, the modified geometry produces a more uniform WSS(cyc) distribution eliminating both high and low WSS(cyc) around the toe, critical in avoiding IT. Peak SWSSG on the artery floor of the modified model is less than one-half that in the conventional case, and high SWSSG at the toe is eliminated. The validated numerical model is useful for modeling unsteady coronary anastomotic flows and elucidating the significance of geometry regulated hemodynamics. The results suggest the clinical relevance of constructing smooth graft-artery transition in distal coronary anastomoses to improve their hemodynamic performance.     

Instability of the two-layered thick-walled esophageal model under the external pressure and circular outer boundary condition

The mucosal folding is a phenomenon observed for some biological tissues, including the pulmonary airway and gastrointestinal tract. In order to understand the mechanism of the formation of mucosal folding, a thick-walled two-layered cylindrical mathematical model was developed to investigate the buckling behavior under the external pressure and circular outer boundary condition. With the finite element method, the validity and accuracy of the proposed model was verified. The results showed that the fold number was in the range of 4-6, which was agreed with the experimental observation for the mucosal folding of a porcine esophagus. The fold number was found to decrease with the increase in the ratio of the inner to outer material stiffness. The increase in the thickness of inner layer also caused a slight declination of the fold number. Since the effects of both the material and geometrical nonlinearities have been accounted for, this model is more general to be used for the prediction of the buckling behavior of the layered structure with a wide range of thickness ratios and/or stiffness ratios.     

Plagiorchis muris infection in Apodemus agrarius from northern Gyeonggi-do (Province) near the demilitarized zone

The small intestines of 6 species of rodents and 1 species of insectivore were examined seasonally for Plagiorchis muris infection in 3 different localities in northern Gyeonggi-do (Province), near the demilitarized zone (DMZ). A total of 1,496 animals, including 1,366 Apodemus agrarius, 54 Crocidura lasiura (insectivore), 32 Mus musculus, 28 Micronytus fortis, 9 Eothenomys regulus, 6 Micronys minutus, and 3 Cricetulus triton, were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804) and Pocheon-gun (343) at 3-mo intervals from December 2004 to September 2005. A total of 1,647 P. muris were collected from 72 (5.3%) A. agrarius. The infection rate was the highest in Pocheon-gun (8.2%), followed by Yeoncheon-gun (5.0%) and Paju-shi (4.2%). A higher infection rate was observed in A. agrarius captured during September (19.4%) than those captured during December (3.0%), June (2.6%), or April (0%). However, the worm burden was the highest in June (av. 32.1/animal), followed by September (24.7), December (4.0), and April (0). None of the other animal species were found infected with P. muris. The results reveal that A. agrarius is a natural definitive host for P. muris, and infection rates and worm burdens vary seasonally and geographically.     

Coupling of glucose response element from L-type pyruvate kinase and G6Pase promoter enhances glucose responsive activity in hepatoma cells

Type 1 diabetes results from the autoimmune destruction of pancreatic β-cells, which leads to severe insulin deficiency. Insulin gene therapy provides an attractive approach to cure diabetes. The critical factor for insulin gene therapy in surrogate cells is to select an appropriate site for insulin expression and a tissue-specific promoter that is responsive to both physiological glucose and insulin concentrations. A novel chimeric promoter, (GIRE)n-G6Pase, consisting of a 1.6 kb glucose 6-phosphatase (G6Pase) promoter and a segment of the regulatory element derived from the L-type pyruvate kinase (L-PK) promoter, was designed to provide strong and tight control of insulin expression in liver. One or three copies of GIRE were linked to the G6Pase promoter, which showed a stronger promoter activity than the G6Pase promoter alone. The chimeric promoter was inhibited by insulin in a dosage-dependent manner and activated by glucose, two features essential for glucose metabolism. The promoter activity is conserved between species and highly specific for liver cells. The construction of a chimeric promoter with stronger and more sensitive responsive activity to glucose and insulin in liver cells could further advance studies in insulin gene therapy. Mr. James Chong was a senior student in the Department of Cell and Molecular Biology, Tulane University. His independent study project was partially overlapped with this study.     

Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C

Spectroscopic methods such as circular dichroism and F?rster resonance energy transfer are current approaches for monitoring protein conformational changes. Those analyses require special equipment and expertise. The need for fluorescence labeling of the protein may interfere with the native structure. We have developed a microtiter plate-based monoclonal antibody (mAb) epitope analysis to detect protein conformational changes in a high throughput manner. This method is based on the concept that the affinity of the antigen-binding site of an antibody for the specific antigenic epitope will change when the 3-D structure of the epitope changes. The effectiveness of this approach was demonstrated in the present study on troponin C (TnC), an allosteric protein in the Ca(2+) regulatory system of striated muscle. Using TnC purified by a highly effective rapid procedure and mAbs developed against epitopes in the N- and C-domains of TnC enzyme-linked immunosorbant assay (ELISA) clearly detected Ca(2+)-induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. On the other hand, Mg(2+)-binding to the C-domain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca(2+)-Mg(2+) exchange at the C-domain metal ion-binding sites. In addition to further understanding of the structure-function relationship of TnC, the data demonstrate that the mAb epitope analysis provides a simple high throughput method for monitoring 3-D structural changes in native proteins under physiological condition and has broad applications in protein structure-function relationship studies.     

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death （PCD）. However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 r_esistant11-1 （/br11-1）, which fails to generate reactive oxygen intermediates （ROIs）, is incapable of initiating PCD when the mutant is challenged by fumonisin B l （FB0, a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 （LCB 1） subunit of serine palmitoyltransferase （SPT）, which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FBl. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine- 1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.     

Activated sludge treatment of a xenobiotic with or without a biogenic substrate during start-up and shocks

Lab-scale continuous flow activated sludge systems that were acclimated to 2,4-dichlorphenoxyacetic acid (2,4-D) under sole 2,4-D influent and without sludge wastage, were able to maintain successful 2,4-D treatment when both 2,4-D and a biogenic substrate were fed and the systems operated with finite mean cell residence times (theta(c)). When the systems were fed dual 2,4-D and biogenic substrates and operated with finite theta(c) from the start, treatment of 2,4-D fluctuated noticeably long after acclimation. At the reintroduction of 2,4-D after its absence from the influent for a period of time (2,4-D shock), the systems under both the sole and dual substrate conditions suffered similar treatment losses; the extent of treatment losses was related to the length of 2,4-D absence time. When shocked, systems with sole 2,4-D influent had a slight advantage over dual substrates by showing a faster recovery from shocks with the help of re-acclimation.     

Docking-based 3D-QSAR study for selectivity of DPP4, DPP8, and DPP9 inhibitors

In order to obtain information regarding the design of selective DPP4 inhibitors, a 3D-QSAR study was conducted using DPP4, DPP8, and DPP9 inhibitors including newly synthesized six- and seven-membered cyclic hydrazine derivatives (KR64300, KR64301), which were evaluated in vitro for their inhibition of DPP4, DPP8, and DPP9. In this study, a highly predictive CoMFA model based on the fast-docking for DPP4, DPP8, and DPP9 inhibitors was obtained. This reliable model showed leave-one-out cross-validation q(2) and conventional r(2) values of 0.68 and 0.96 for the DPP4 inhibitors, 0.58 and 0.98 for the DPP8 inhibitors, and 0.68 and 0.97 for the DPP9 inhibitors, respectively. The validation of the CoMFA model was confirmed by the compounds in the test set, including the synthesized six- and seven-membered cyclic hydrazines. According to this study, to obtain selective DPP4 inhibitors compared to their isozymes, the interaction of the inhibitors with the S3 site and S1' site in DPP4 must be considered. The proposed newly synthesized compounds, KR64300 and KR64301, interact well with the sites mentioned above, showing excellent selectivity.     

西双版纳热带雨林凋落叶分解过程Ⅱ.微生物与线虫的群落动态

通过野外试验与室内模拟相结合,对西双版纳热带雨林生态系统混合凋落叶分解过程中微生物和线虫种群动态变化进行了系统研究。野外试验采用网袋法（即1mm和100μm网眼网袋,分别限制大型土壤动物和螨类的进入）,室内试验采用灭菌一接种法,以观测不同生物组成条件下线虫和微生物对凋落叶的分解作用。结果表明,伴随分解进程,微生物基本呼吸速率不断下降且与分解进程正相关。利用底物诱导呼吸法测定微生物生物量以及细菌、真菌生物量的变化表明,微生物在凋落叶分解过程中的演替遵循一定的路线。当土壤动物参与分解时,由于捕食压力的存在,微生物一般按照“双峰”型路线变化,并存在明显的生物演替现象,在这个过程中微生物对C的利用效率也不断提高;当不存在这种捕食压力时,微生物表现为“单峰-递减”式发展模式,生物量由强到弱变化,微生物对C的利用效率持续下降。土壤动物在凋落叶分解过程中表现为“单峰”型变化动态,与微生物量“双峰”动态形成互补,捕食者与被捕食者之间是一种“捕食-激发”作用下的种群消长关系,这种关系的强烈程度与捕食压力有关。“双峰”发展路线在一定程度上加速了凋落叶的分解进程。