Biomedical subjects

Wen Liu

Publications and source records attributed to Wen Liu.

At least 37 records · Page 2Linked to original sources

PubMed2006

Role of Tyr348 in Tyr385 radical dynamics and cyclooxygenase inhibitor interactions in prostaglandin H synthase-2.

Both prostaglandin H synthase (PGHS) isoforms utilize a radical at Tyr385 to abstract a hydrogen atom from arachidonic acid, initializing prostaglandin synthesis. A Tyr348-Tyr385 hydrogen bond appears to be conserved in both isoforms; this hydrogen bonding has the potential to modulate the positioning and reactivity of the Tyr385 side chain. The EPR signal from the Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in both isoforms. In PGHS-2, this transition results from radical migration from Tyr385 to Tyr504. Localization of the radical to Tyr385 in the recombinant human PGHS-2 Y504F mutant was exploited in examining the effects of blocking Tyr385 hydrogen bonding by introduction of a further Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in the Y348F/Y504F mutant, but the Tyr385 radical was formed more slowly and had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in the parent Y504F mutant. The effect of disrupting Tyr385 hydrogen bonding on the cyclooxygenase active site structure was probed by examination of cyclooxygenase inhibitor kinetics. Aspirin treatment eliminated all oxygenase activity in the Y348F/Y504F double mutant, with no indication of the lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of the Y348F mutation also strengthened the time-dependent inhibitory action of nimesulide. These results suggest that removal of Tyr348-Tyr385 hydrogen bonding in PGHS-2 allows greater conformational flexibility in the cyclooxygenase active site, resulting in altered interactions with inhibitors and altered Tyr385 radical behavior.

Amino Acid Substitution↗

PubMed2006

Proteome comparison following self- and across-pollination in self-incompatible apricot (Prunus armeniaca L.).

The study compared the protein differences between self- and across-pollinated self-incompatible (SI) apricots by two-dimensional gel electrophoresis and liquid chromatography-electrospray ion trap tandem mass spectrometry, the results showed that nine protein spots were expressed in self-pollinated pistil and only one was expressed in cross-pollinated pistils. Sixteen and three protein spots were up- and down-regulated in cross-pollinated pistils, respectively, compared with self-pollinated pistils. Seven protein spots were identified unambiguously by SEQUEST in NCBI protein database: Actin-12, enolase, MYB transcription-factor-like protein, heat-shock protein 70 were upregulated in cross-pollinated pistils compared with self-pollinated pistils; and actin-7, actin-8 and fructose bisphosphate aldolase-like protein were detected only in self-pollinated pistils.

Amino Acid Sequence↗

PubMed2006

Detection and transcript expression of S-RNase gene associated with self-incompatibility in apricot (Prunus armeniaca L.).

The identity and expression of S-RNase genotypes in the self-compatible (SC) apricot cultivar 'Katy' and the self-incompatible (SI) cultivar 'Xinshiji' were examined. We used allele specific polymerase chain reaction (AS-PCR) and designated the alleles in 'Katy' and 'Xinshiji' as S(8)Sc and S(9)S(10), respectively. The S-RNase gene was expressed in style at the balloon stage in both genotypes. Using real-time fluorescence quantification RT-PCR technology (FQRT-PCR), spatio-temporal expression patterns of S-RNase gene between 'Katy' and 'Xinshiji' were compared. The results revealed that the expression of the S-RNase gene in 'Katy' and 'Xinshiji' were different. The transcript abundance was distinctly diverse at the key stage (i.e., at 24 h after self-pollination) in both genotypes, and was greater in 'Xinshiji' (SI) than 'Katy' (SC). In addition, the abundance of the S-RNase transcript was higher in upper-half of style than in the lower-half of style or in the ovary. In the SI cultivar 'Xinshiji', the expression of S-RNase reminded a relatively high level after cross-pollination, but it dropped continuously after self-pollination and un-pollination.

Alleles↗

PubMed2006

Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis.

The biosynthetic gene cluster for chlorothricin (CHL) was localized to a 122 kb contiguous DNA from Streptomyces antibioticus DSM 40725, and its involvement in CHL biosynthesis was confirmed by gene inactivation and complementation. Bioinformatic analysis of the sequenced 111.989 kb DNA region revealed 42 open reading frames, 35 of which were defined to constitute the CHL gene cluster. An assembly model for CHL biosynthesis from D-olivose, 2-methoxy-5-chloro-6-methylsalicyclic acid, and chlorothricolide building blocks was proposed. This work represents cloning of a gene cluster for spirotetronate antibiotic biosynthesis and sets the stage to investigate the unusual macrolide biosynthesis including tandem Diels-Alder cyclizations, Baeyer-Villiger oxidation, and incorporation of an enoylpyruvate unit.

Amino Acid Sequence↗

PubMed2006

Divergent cyclooxygenase responses to fatty acid structure and peroxide level in fish and mammalian prostaglandin H synthases.

Prostanoid synthesis in mammalian tissues is regulated at the level of prostaglandin H synthase (PGHS) cyclooxygenase catalysis by the availability and structure of substrate fatty acid and the availability of peroxide activator. Two major PGHS isoforms, with distinct pathophysiological functions and catalytic regulation, have been characterized in mammals; a functionally homologous PGHS isoform pair has been cloned from an evolutionarily distant vertebrate, brook trout. The cyclooxygenase activities of recombinant brook trout PGHS-1 and -2 were characterized to test the generality of mammalian regulatory paradigms for substrate specificity, peroxide activation, and product shifting by aspirin. Both trout cyclooxygenases had much more restrictive substrate specificities than their mammalian counterparts, with pronounced discrimination toward arachidonate (20:4n-6) and against eicosapentaenoate (20:5n-3) and docosahexaenoate (22:6n-3), the latter two prominent in trout tissue lipids. Aspirin treatment did not increase lipoxygenase-type catalysis by either trout enzyme. Both trout enzymes had higher requirements for peroxide activator than their mammalian counterparts, though the preferential peroxide activation of PGHS-2 over PGHS-1 seen in mammals was conserved in the fish enzymes. The divergence in cyclooxygenase characteristics between the trout and mammalian PGHS proteins may reflect accomodations to differences among vertebrates in tissue lipid composition and general redox state.

Animals↗

PubMed2006

[Preparation of recombinant IL-1 fusion protein as the substrate for HCV NS3 serine protease].

AIM: To prepare recombinant IL-1 fusion protein as the substrate for the HCV NS3 serine protease. METHODS: NS5A-B gene fragment (2,412-2,427aa) synthesized by PCR was subcloned into prokaryotic expression vector pBVIL1 to fuse with IL-1 gene and the recombinant vector pBVIL1/NS5A-B was transformed into E.coli strain HB101. The fused protein was induced to express at 42degreesCelsius and purified by two-step column chromatography. The proteolysis of the purified IL-1 fusion protein catalyzed by NS3 serine protease was analyzed with SDS-PAGE, Western blot and ELISA. RESULTS: NS5A-B fragment gene was correctly subcloned into pBVIL1 vector and the fusion protein was expressed as inclusion body in transformed HB101 cells. The recombinant fusion protein can be cleaved into smaller fragments by NS3 protease. CONCLUSION: The recombinant fusion protein can be cleaved by NS3 serine protease successfully and specifically, suggesting that it can be used as a surrogate substrate of NS3 serine protease in searching for inhibitors of this protease.

Amino Acid Sequence↗

PubMed2006

[Animal model of facial neuritis induced by herpes simplex virus].

OBJECTIVE: To study the role of herpes simplex virus type 1 ( HSV-1 ) in facial paralysis by developing an experimental animal model of viral facial paralysis. METHODS: Both sides of posterior auricular branch of facial nerve were anatomies and incised in 66 mice. The HSV-1 was inoculated into right ear branch and fetal bovine serum was inoculated into left ear branch as control. The symmetry of mouse face was observed and scored. The temporal bones were serially sectioned and stained with hematoxylin and eosin. The extratemporal facial nerves were stained with osmium tetroxide. HSV-1 DNA in bilateral facial nerve, brain stem, trigeminal ganglion and spinal cord was detected by the polymerase chain reaction. RESULTS: Twenty-eight (42. 42%) mice developed right facial paralysis between 2 and 5 days after inoculation. Continuing 3-6 days, the facial paralysis recovered spontaneously. Thirty-eight mice had no signs of facial paralysis. Compared with the left, nerve swelling, inflammatory cell infiltration were manifested in right temporal facial nerve of paralyzed mice. The ratio of the cross-sectional area of the facial nerve to the facial canal ( FN/FC ) was significantly higher than that on the control side (P < 0.01). Demyelinated nerve fibers were seen in the right extratemporal facial nerve. Not only in paralyzed mice, but also in non-paralyzed mice, HSV DNA was detected in some nerve tissues. CONCLUSIONS: Inoculating HSV-1 into posterior auricular branch of facial nerve can produce an acute and transient facial paralysis in mice. The possible pathophysiologic mechanism of the facial paralysis is viral invasion and transportation from distal branch to main trunk. Then the viral facial neuritis causes facial paralysis.

Animals↗

PubMed2006

[Effect of three flavones on enzyme activity of recombinant human phosphoinositide 3-kinase p110beta catalytic subunit].

OBJECTIVE: To study the effect of three flavones-luteolin, apigenin and genistein on activity of recombinant human phosphoinositide 3-kinase (PI3-K) p110beta catalytic subunit. METHODS: Recombinant human P13-K p110beta catalytic subunit was expressed by gene engineering. PI3-K activity was assayed by incubation recombinant PI3-K p110beta with phosphatidylinostiol-4,5-bisphosphate and [gamma-32P] ATP; the 32P-radiolabeled lipids were extracted with cholroform and methanol, and assessed by scintillation counter. RESULTS: Luteolin and apigenin showed inhibition on the recombinant p110beta catalytic subunit with IC50 8. 65 micromol/L and 11.56 micromol/L, but genistein had no inhibition. CONCLUSION: Luteolin and apigenin are inhibitors of P13-K. The recombinant P13-K p1100 catalytic subunit may be used as a molecular target for simpler screening and development of more effective inhibitors of P13-K.

Apigenin↗

PubMed2006

Performance of hybrid constructed wetland systems for treating septic tank effluent.

The integrated wetland systems were constructed by combining horizontal-flow and vertical-flow bed, and their purification efficiencies for septic tank effluent were detected when the hydraulic retention time (HRT) was 1 d, 3 d, 5 d under different seasons. The results showed that the removal efficiencies of the organics, phosphorus were steady in the hybrid systems, but the removal efficiency of total nitrogen was not steady due to high total nitrogen concentration in the septic tank effluent. The average removal rates of COD (chemical oxygen demand) were 89%, 87%, 83%, and 86% in summer, autumn, winter and spring, respectively, and it was up to 88%, 85%, 73%, and 74% for BOD5 (5 d biochemical oxygen demand) removal rate in four seasons. The average removal rates of TP (total phosphorous) could reach up to 97%, 98%, 95%, 98% in four seasons, but the removal rate of TN (total nitrogen) was very low. The results of this study also indicated that the capability of purification was the worst in winter. Cultivating with plants could improve the treated effluent quality from the hybrid systems. The results of the operation of the horizontal-flow and vertical-flow cells (hybrid systems) showed that the removal efficiencies of the organics, TP and TN in horizontal-flow and vertical-flow cells were improved significantly with the extension of HRT under the same season. The removal rate of 3 d HRT was obviously higher than that of 1 d HRT, and the removal rate of 5 d HRT was better than that of 3 d HRT, but the removal efficiency was not very obvious with the increment of HRT. Therefore, 3 d HRT might be recommended in the actual operation of the hybrid systems for economic and technical reasons.

Biodegradation, Environmental↗

PubMed2006

Trunk rotational strength training for the management of adolescent idiopathic scoliosis (AIS).

Quantified trunk rotational strength training has shown promise as a non-operative management option for individuals with AIS. The purposes of our study are to test whether a quantified trunk rotational strength training protocol can increase trunk strength and stabilize or decrease curve size. Seven adolescents with AIS (5 female 2 male; mean 14 yrs +/- 2.6 yrs; mean Cobb 28 degrees +/- 6 degrees range 20 degrees -37 degrees) underwent four months of supervised trunk rotational strength training, and repeat strength test. Trunk strength in both directions increased significantly after training (p<0.05). Average Cobb angle decreased to 23 degrees +/- 11 degrees (range 6 degrees -35 degrees). Four individuals showed reduction (>5 degrees) in their original curve, and 3 remained the same (+/-5 degrees). The strength training protocol significantly increased isometric rotational strength and scoliosis was stabilized short term.

Adolescent↗

PubMed2006

Comparison of isometric trunk rotational strength of adolescents with idiopathic scoliosis to healthy adolescents.

Trunk rotational strength asymmetry has been suggested in adolescents with idiopathic scoliosis (IS), but is unconfirmed. The sitting isometric trunk rotational strength, at neutral and 18 degrees or 36 degrees of right or left pre-rotation, of a group of healthy adolescent females (CG), n=12, is compared with a group of female adolescents with IS (ISG), n= 14. Torque values were normalized to lean body weight. There is a significant weakness when rotating towards the concavity found in patients with AIS at the 36 lo (p 0.07 marginal), 18 lo (p<0.03) and neutral positions (p<0.02), with no side strength asymmetry found in a cohort of healthy adolescents without AIS.

Adolescent↗

PubMed2006

A new microsurgical technique to correct retinal detachment.

PURPOSE: To evaluate the efficacy of a new technique to repair retinal detachments (RD) under the microscope. METHODS: Thirty-six consecutive patients (36 eyes) who presented to our clinic with rhegmatogenous RD without severe proliferative vitreoretinopathy (< or =C1) were included. The sutures for buckling and/or encircling bands were preplaced according to the preoperative location of the breaks using a three-mirror contact lens. Drainage of subretinal fluid, retinal cryotherapy, buckling, locating the retinal breaks, and intravitreal gases injection were performed under surgical microscopy. The surgical effects were compared with those in 37 consecutive patients with rhegmatogenous RD who underwent surgery under binocular indirect ophthalmoscopy. RESULTS: The simultaneous intraoperative observation of fundus details and the sclera through the microscope was excellent in all cases. The effect of retinal cryotherapy was clearly visible. Mild opacity of the refractive media did not interfere with observing cryotherapy and locating the breaks. Retinal reattachment was obtained in 31 eyes (86%) during the primary surgery and in three eyes after a second surgery (94% total). The best-corrected visual acuity was < 0.1 in 6 eyes (16.7%), 0.1-0.4 in 15 eyes (41.7%) and > or = 0.5 in 15 eyes (41.7%). The results were similar to that of RD surgery performed under indirect ophthalmoscopy. CONCLUSIONS: This microsurgical procedure to correct RD is simple, convenient, reliable, provides an upright image, and facilitates good recovery similar to conventional RD surgery.

Adolescent↗

PubMed2005

Heightened epithelial Na+ channel-mediated Na+ absorption in a murine polycystic kidney disease model epithelium lacking apical monocilia.

The Tg737 degrees (rpk) autosomal recessive polycystic kidney disease (ARPKD) mouse carries a hypomorphic mutation in the Tg737 gene. Because of the absence of its protein product Polaris, the nonmotile primary monocilium central to the luminal membrane of ductal epithelia, such as the cortical collecting duct (CCD) principal cell (PC), is malformed. Although the functions of the renal monocilium remain elusive, primary monocilia or flagella on neurons act as sensory organelles. Thus we hypothesized that the PC monocilium functions as a cellular sensor. To test this hypothesis, we assessed the contribution of Polaris and cilium structure and function to renal epithelial ion transport electrophysiology. Properties of Tg737 degrees (rpk) mutant CCD PC clones were compared with clones genetically rescued with wild-type Tg737 cDNA. All cells were grown as polarized cell monolayers with similarly high transepithelial resistance on permeable filter supports. Three- to fourfold elevated transepithelial voltage (V(te)) and short-circuit current (I(sc)) were measured in mutant orpk monolayers vs. rescued controls. Pharmacological and cell biological examination of this enhanced electrical end point in mutant monolayers revealed that epithelial Na(+) channels (ENaCs) were upregulated. Amiloride, ENaC-selective amiloride analogs (benzamil and phenamil), and protease inhibitors (aprotinin and leupeptin) attenuated heightened V(te) and I(sc). Higher concentrations of additional amiloride analogs (ethylisopropylamiloride and dimethylamiloride) also revealed inhibition of V(te). Cell culture requirements and manipulations were also consistent with heightened ENaC expression and function. Together, these data suggest that ENaC expression and/or function are upregulated in the luminal membrane of mutant, cilium-deficient orpk CCD PC monolayers vs. cilium-competent controls. When the genetic lesion causes loss or malformation of the monocilium, ENaC-driven Na(+) hyperabsorption may explain the rapid emergence of severe hypertension in a majority of patients with ARPKD.

Amiloride↗

PubMed2005

Biosynthesis of the beta-amino acid moiety of the enediyne antitumor antibiotic C-1027 featuring beta-amino acyl-S-carrier protein intermediates.

The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. The biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety (boxed) of the C-1027 chromophore (1) from l-tyrosine (3) and its incorporation into 1 are catalyzed by six enzymes: SgcC, SgcC1, SgcC2, SgcC3, SgcC4, ShcC5. In vivo and in vitro characterization of these enzymes delineated this pathway, unveiling a novel strategy for beta-amino acid modification featuring beta-amino acyl-S-carrier protein intermediates. These findings shed new light into beta-amino acid biosynthesis and present a new opportunity to engineer the C-1027 biosynthetic machinery for the production of novel analogues as exemplified by 20-deschloro-C-1027 (4), 20-deschro-22-deshydroxy-C-1027 (5), and 22-deshydroxy-C-1027 (6).

Acyl Carrier Protein↗

PubMed2005

Focal adhesion kinase plays a pivotal role in herpes simplex virus entry.

Development of strategies to prevent herpes simplex virus (HSV) infection requires knowledge of cellular pathways harnessed by the virus for invasion. This study demonstrates that HSV induces rapid phosphorylation of focal adhesion kinase (FAK) in several human target cells and that phosphorylation is important for entry post-binding. Nuclear transport of the viral tegument protein VP16, transport of viral capsids to the nuclear pore, and downstream events (including expression of immediate-early genes and viral plaque formation) were substantially reduced in cells transfected with dominant-negative mutants of FAK or small interfering RNA designed to inhibit FAK expression. These observations were substantiated using mouse embryonic fibroblast cells derived from embryonic FAK-deficient mice. Infection was reduced by >90% in knockout cells relative to control cells and was further reduced if the knockout cells were transfected with small interfering RNA targeting proline-rich tyrosine kinase-2, which was also phosphorylated in response to HSV. The knockout cells were permissive for viral binding, and virus triggered an intracellular calcium response, but nuclear transport was inhibited. Together, these results support a novel model for invasion that implicates FAK phosphorylation as important for delivery of viral capsids to the nuclear pore.

Active Transport, Cell Nucleus↗

PubMed2005

Mechanoregulation of intracellular Ca2+ concentration is attenuated in collecting duct of monocilium-impaired orpk mice.

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the progressive dilatation of collecting ducts, the nephron segments responsible for the final renal regulation of sodium, potassium, acid-base, and water balance. Murine models of ARPKD possess mutations in genes encoding cilia-associated proteins, including Tg737 in orpk mice. New findings implicate defects in structure/function of primary cilia as central to the development of polycystic kidney disease. Our group (Liu W, Xu S, Woda C, Kim P, Weinbaum S, and Satlin LM, Am J Physiol Renal Physiol 285: F998-F1012, 2003) recently reported that increases in luminal flow rate in rabbit collecting ducts increase intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells therein. We thus hypothesized that fluid shear acting on the apical membrane or hydrodynamic bending moments acting on the cilium increase renal epithelial [Ca(2+)](i). To further explore this, we tested whether flow-induced [Ca(2+)](i) transients in collecting ducts from mutant orpk mice, which possess structurally abnormal cilia, differ from those in controls. Isolated segments from 1- and 2-wk-old mice were microperfused in vitro and loaded with fura 2; [Ca(2+)](i) was measured by digital ratio fluorometry before and after the rate of luminal flow was increased. All collecting ducts responded to an increase in flow with an increase in [Ca(2+)](i), a response that appeared to be dependent on luminal Ca(2+) entry. However, the magnitude of the increase in [Ca(2+)](i) in 2- but not 1-wk-old mutant orpk animals was blunted. We speculate that this defect in mechano-induced Ca(2+) signaling in orpk mice leads to aberrant structure and function of the collecting duct in ARPKD.

Animals↗

PubMed2005

Broadband polarizing beam splitter with an embedded metal-wire nanograting.

An embedded metal-wire nanograting is designed and fabricated for the first time to our knowledge. This artificial material could be used as a broadband polarizing beam splitter to reflect s-polarized light and transmit p-polarized light. An upper-cladding layer of the same material as the gratings is deposited on the ridge of the gratings, whereas the metal wire is deposited in the grating trenches. This embedded structure makes the grating more firm in its applications. High polarization efficiency and low insertion loss with a broad wavelength range (900-1700 nm) and a wide angular tolerance are obtained by optimization of the designed structure.

Journal Article↗

PubMed2005

Dietary K+ regulates apical membrane expression of maxi-K channels in rabbit cortical collecting duct.

The cortical collecting duct (CCD) is a final site for regulation of K(+) homeostasis. CCD K(+) secretion is determined by the electrochemical gradient and apical permeability to K(+). Conducting secretory K(+) (SK/ROMK) and maxi-K channels are present in the apical membrane of the CCD, the former in principal cells and the latter in both principal and intercalated cells. Whereas SK channels mediate baseline K(+) secretion, maxi-K channels appear to participate in flow-stimulated K(+) secretion. Chronic dietary K(+) loading enhances the CCD K(+) secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol 104: 693-710, 1994). Long-term exposure of Ambystoma tigrinum to elevated K(+) increases renal K(+) excretion due to an increase in apical maxi-K channel density in their CDs (Stoner and Viggiano, J Membr Biol 162: 107-116, 1998). The purpose of the present study was to test whether K(+) adaptation in the mammalian CCD is associated with upregulation of maxi-K channel expression. New Zealand White rabbits were fed a low (LK), control (CK), or high (HK) K(+) diet for 10-14 days. Real-time PCR quantitation of message encoding maxi-K alpha- and beta(2-4)-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals (P < 0.05); beta(1)-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of alpha-subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in alpha-intercalated cells in HK kidneys. In summary, K(+) adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical alpha-subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane.

Amino Acid Sequence↗