Protective Effect of Eupatilin Pretreatment Against Hepatic Ischemia-Reperfusion Injury in Mice.

BACKGROUND: Eupatilin, a pharmacologically active flavone derived from Artemisia species, is known to have antioxidant and antiinflammatory activities. Ischemia-reperfusion injury (IRI) is a major critical event that commonly occurs after liver transplantation and resection. Furthermore, inflammatory responses to IRI exacerbate the resultant hepatic injury. In this study, we investigated whether eupatilin protects against IR-induced acute liver injury in mice. MATERIALS AND METHODS: Partial (70%) hepatic IRI was induced in male C57BL/6 mice by portal triad pedicle occlusion for 90 minutes followed by reperfusion for 6 hours. Eupatilin (10 mg/kg body weight, oral) was administered 4 days before the IRI. RESULTS: Treatment with eupatilin significantly decreased serum alanine aminotransferase and serum aspartate aminotransferase as well as liver histologic changes. Eupatilin also prevented hepatic glutathione depletion and increased malondialdehyde levels induced by IRI. Western blotting indicated that eupatilin significantly increased the levels of heat shock protein and B-cell lymphoma 2 protein, attenuated inducible nitric oxide synthase, and cleaved caspase-3 levels 6 hours after IRI. The expression of the Toll-like receptor 2/4, and phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor was significantly decreased in the eupatilin pretreatment group. CONCLUSIONS: Eupatilin improved the acute hepatic IRI by reducing inflammation and apoptosis. These findings suggest that eupatilin is a promising therapeutic agent against acute IR-induced hepatic damage.

5,7-dihydroxy-3,4,6-trimethoxyflavone attenuates ischemic damage and apoptosis in mouse islets.

BACKGROUND: The transplantation of isolated pancreatic islets is a promising treatment for diabetes. 5,7-dihydroxy-3,4,6-trimethoxyflavone (Eupatilin), a pharmacologically active flavone derived from the Artemisia plant species, has been reported to have antioxidant and anti-inflammatory activities. This study examines the hypothesis that preoperative eupatilin treatment can attenuate ischemic damage and apoptosis before islet transplantation. METHODS: Islets isolated from Balb/c mice were randomly divided into 2 groups, and cultured in medium supplemented with or without eupatilin. In vitro islet viability and function were assessed. After treatment with a cytokine cocktail consisting of tumor necrosis factor (TNF)-α, interferon (INF)-γ, and interleukin (IL)-1ß, islet cell viability, function, and apoptotic status were determined. The glutathione (GSH) and nitrous oxide (NO) levels were also measured. Proteins related to apoptosis were analyzed using Western blotting. RESULTS: There was no difference in cell viability between the 2 groups. Islets cultured in the medium supplemented with eupatilin showed 1.4-fold higher glucose-induced insulin secretion than the islets cultured in the medium without eupatilin. After treatment with a cytokine cocktail, glucose-induced insulin release and the total insulin content of the islets were significantly improved in eupatilin-pretreated islets compared with islets not treated with eupatilin. Apoptosis was significantly decreased, and GSH levels were elevated in the eupatilin-pretreated group. Cytokine-only treated islets produced significantly higher levels of NO, iNOS, and caspase-3 than islets pretreated with eupatilin before cytokine treatment. CONCLUSIONS: These results suggest that preoperative eupatilin administration enhances islet function before transplantation and attenuates the cytokine-induced damage associated with NO production and apoptosis.

Protective effect of eupatilin against renal ischemia-reperfusion injury in mice.

BACKGROUND: Eupatilin, a pharmacologically active flavone derived from Artemisia species, is known to have antioxidant and anti-inflammatory activities. Ischemia-reperfusion injury (IRI) is a major complication after renal transplantation, with inflammatory responses to IRI exacerbating the resultant renal injury. In the present study, we investigated whether eupatilin exhibits renoprotective activities against ischemia-reperfusion-induced acute kidney injury in mice. MATERIALS AND METHODS: Renal IRI was induced in male C57BL/6 mice by bilateral renal pedicle occlusion for 30 minutes followed by reperfusion for 48 hours. Eupatilin (10 mg/kg body weight p.o.) was administered 4 days before IRI. RESULTS: Treatment with eupatilin significantly decreased neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels in urine, blood urea nitrogen level, and serum creatinine levels, as well as kidney tubular injury. Western blotting indicated that eupatilin significantly increased the levels of heat shock protein 70 and B-cell lymphoma protein, and it attenuated inducible nitric oxide synthase, Bcl-2-associated X protein, and caspase-3 levels 48 hours after IRI. CONCLUSION: Our findings suggest that eupatilin is a promising therapeutic agent against acute ischemia-induced renal damage.

Scleral fixation of Ahmed glaucoma valve tube tip for adjustment of cornea-touching malposition.

PURPOSE: Tube-corneal touch occurring after Ahmed glaucoma valve (AGV) implantation is conventionally treated by tube cutting or tube transposition from the original pathway. However, in some cases, tube cutting is insufficient, and rearranging the pathway of the tube through a new sclera tunnel, ciliary sulcus, or pars plana is not feasible, as the conjunctiva and sclera covering the tube are difficult to be redissected. So, we propose a novel technique that repositions malpositioned AGV tube using scleral fixation and its successful applications in two patients with tube-corneal touch. METHODS: (A) A scleral flap is made at the point for scleral fixation. (B) The anterior chamber is maintained using an anterior chamber maintainer. The incision is made immediately above the tube entering the anterior chamber and the tube end is flipped out using a Sinskey. (C) A double-armed 10/0 prolene straight needle is penetrated through the tube end. The leading needle enters the anterior chamber through the previously made incision and is pulled through the scleral flap. (D) The tube tip and the second needle of the double-armed 10/0 prolene straight needle also enter the anterior chamber through the previously made incision and the second needle is pulled through the scleral flap. The tube end is extended to be parallel to the cornea surface. RESULTS: Patients maintained good tube positioning without any serious complications during average of 15 months of follow-up after operation. CONCLUSION: We believe that our method is a simple and minimally invasive surgical method for treating AGV tube touching of the corneal endothelium. However, considering the limited number of cases studied and the short follow-up period, a larger group with a longer follow-up period is necessary.

Indepth diagnosis of a secondary clarifier by the application of radiotracer technique and numerical modeling.

To make an indepth diagnosis of a full-scale rectangular secondary clarifier, an experimental and numerical study has been performed in a wastewater treatment facility. Calculation results by the numerical model with the adoption of the SIMPLE algorithm of Patankar are validated with radiotracer experiments. Emphasis is given to the prediction of residence time distribution (RTD) curves. The predicted RTD profiles are in good agreement with the experimental RTD curves at the upstream and center sections except for the withdrawal zone of the complex effluent weir structure. The simulation results predict successfully the well-known flow characteristics of each stage such as the waterfall phenomenon at the front of the clarifier, the bottom density current and the surface return flow in the settling zone, and the upward flow in the exit zone. The detailed effects of density current are thoroughly investigated in terms of high SS loading and temperature difference between influent and ambient fluid. The program developed in this study shows the high potential to assist in the design and determination of optimal operating conditions to improve effluent quality in a full-scale secondary clarifier.

Study of flow characteristics in a secondary clarifier by numerical simulation and radioisotope tracer technique.

Numerical simulation in a 2-D rectangular coordinate and experimental study have been performed to figure out the flow characteristics and concentration distribution of a large-scale rectangular final clarifier in wastewater treatment facility located in Busan, S. Korea. The purpose of numerical calculation is to verify the experimentally measured data by radioisotope tracer technique and further to understand the important physical feature occurring in a large-scale clarifier, in many cases which is not sufficient by the aid of limited number of experimental data. To this end, a comprehensive computer program is basically made by SIMPLE algorithm by Patankar with the special emphasis on the parametric evaluation of the various phenomenological models. Calculation results are successfully evaluated against experimental data obtained by the method of radioisotope tracer. Detailed comparison is made on the calculated residence time distribution (RTD) curves with measurement inside the clarifier as well as the exhaust. Further the calculation results predict well the well-known characteristics of clarifier flow such as the waterfall phenomenon at the front end of the clarifier, the bottom density current in the settling zone and the upward flow in the withdrawal zone. Thus it is believed that the flow calculation program and the data incorporation technique of radioisotope measurement employed in this study show the high possibility as a complementary tool of experiment in this area.

Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.

A new guaianolide as apoptosis inhibitor from Chrysanthemum boreale.

The 8-O-acetyl-3,10-dihydroxy-4(15),11(13)-guaiadien-12,6-olide, named borenolide, was isolated from Chrysanthemum boreale M. Borenolide inhibits the etoposide-induced apoptosis in U937 cell with an IC(50) value of 6.2 microg/ml. Structural assignment was based on NMR-spectroscopic methods and X-ray crystallographic analysis.

15N NMR relaxation studies of backbone dynamics in free and steroid-bound Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni.

The backbone dynamics of Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S(2), tau(e), and R(ex)) and the overall rotational correlation time (tau(m)). The rotational correlation times were 19.23 +/- 0.08 and 17.08 +/- 0.07 ns with the diffusion anisotropies (D( parallel)/D( perpendicular)) of 1.26 +/- 0.03 and 1.25 +/- 0.03 for the free and steroid-bound KSI, respectively. The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in helix A1 and strand B4. However, the majority of the residues exhibit reduced order parameters upon ligand binding. In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme. Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding.

Folding mechanism of ketosteroid isomerase from Comamonas testosteroni.

Ketosteroid isomerase (KSI) from Comamonas testosteroni is a homodimeric enzyme with 125 amino acids in each monomer catalyzing the allylic isomerization reaction at rates comparable to the diffusion limit. Kinetic analysis of KSI refolding has been carried out to understand its folding mechanism. The refolding process as monitored by fluorescence change revealed that the process consists of three steps with a unimolecular fast, a bimolecular intermediate, and most likely unimolecular slow phases. The fast refolding step might involve the formation of structured monomers with hydrophobic surfaces that seem to have a high binding capacity for the amphipathic dye 8-anilino-1-naphthalenesulfonate. During the refolding process, KSI also generated a state that can bind equilenin, a reaction intermediate analogue, at a very early stage. These observations suggest that the KSI folding might be driven by the formation of the apolar active-site cavity while exposing hydrophobic surfaces. Since the monomeric folding intermediate may contain more than 83% of the native secondary structures as revealed previously, it is nativelike taking on most of the properties of the native protein. Urea-dependence analysis of refolding revealed the existence of folding intermediates for both the intermediate and slow steps. These steps were accelerated by cyclophilin A, a prolyl isomerase, suggesting the involvement of a cis-trans isomerization as a rate-limiting step. Taken together, we suggest that KSI folds into a monomeric intermediate, which has nativelike secondary structure, an apolar active site, and exposed hydrophobic surface, followed by dimerization and prolyl isomerizations to complete the folding.

Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B.

Equilibrium and kinetic analyses have been performed to elucidate the roles of dimerization in folding and stability of KSI from Pseudomonas putida biotype B. Folding was reversible in secondary and tertiary structures as well as in activity. Equilibrium unfolding transition, as monitored by fluorescence and ellipticity measurements, could be modeled by a two-state mechanism without thermodynamically stable intermediates. Consistent with the two-state model, one dimensional (1D) NMR spectra and gel-filtration chromatography analysis did not show any evidence for a folded monomeric intermediate. Interestingly enough, Cys 81 located at the dimeric interface was modified by DTNB before unfolding. This inconsistent result might be explained by increased dynamic motion of the interface residues in the presence of urea to expose Cys 81 more frequently without the dimer dissociation. The refolding process, as monitored by fluorescence change, could best be described by five kinetic phases, in which the second phase was a bimolecular step. Because <30% of the total fluorescence change occurred during the first step, most of the native tertiary structure may be driven to form by the bimolecular step. During the refolding process, negative ellipticity at 225 nm increased very fast within 80 msec to account for >80% of the total amplitude. This result suggests that the protein folds into a monomer containing most of the alpha-helical structures before dimerization. Monitoring the enzyme activity during the refolding process could estimate the activity of the monomer that is not fully active. Together, these results stress the importance of dimerization in the formation and maintenance of the functional native tertiary structure.

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni.

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.

Two cases of chronic pancreatitis with pseudocyst complicated by obstructive jaundice.

We recently treated two cases of chronic pancreatitis with obstructive jaundice due to compression of the common bile duct by pancreatic pseudocyst. The two cases were males admitted with the complaint of icteric skin color. The first, a 46-year-old male, admitted with the complaint of icteric skin color. He was treated by operative cystojejunostomy after percutaneous drainage of the pseudocyst and percutaneous transhepatic biliary drainage. The other case was a 58 year-old male who admitted with the complaint of icteric skin color. He had an infected pseudocyst in the pancreas and was endoscopically treated. Both of them were discharged with favorable clinical course and normal laboratory findings after the treatment. The former patient remained well 11 months after treatment, but the latter patient died from necrotizing pancreatitis and septic shock 6 months after treatment. Most cases of obstructive jaundice associated with pseudocysts appear to be due to fibrotic stricture of the intrapancreatic portion of the common bile duct rather than due to compression of the bile duct by the pseudocyst. In a patient with secondary pancreatic infection or obstructive jaundice following pancreatic disease, differentiating between these two conditions is an important aspect of accurate diagnosis and therapy. Herein we report two unusual cases of chronic pancreatitis with pseudocyst complicated by obstructive jaundice.

Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.

Flavonol glycoside gallate and ferulate esters from Persicaria lapathifolia as inhibitors of superoxide production in human monocytes stimulated by unopsonized zymosan.

Aerial parts of Persicaria lapathifolia S.F. Gray (Polygonaceae) exhibited an inhibitory effect on superoxide production in unopsonized zymosan-stimulated human monocytes. Two known compounds, quercetin 3-O-beta-(2"-galloyl)-glucopyranoside and quercetin 3-O-beta-(2"-galloyl)-rhamnopyranoside, and a new compound, quercetin 3-O-beta-(6"-feruloyl)-galactopyranoside, were isolated as the inhibitors of superoxide production by activity-guided fractionation. IC50 values were shown at the concentrations of 2.1 microM by quercetin 3-O-beta-(2"-galloyl)-glucopyranoside, 1.9 microM by quercetin 3-O-beta-(2"-galloyl)-rhamnoypranoside, and 3.5 microM by quercetin 3-O-beta-(6"-feruloyl)-galactopyranoside whose inhibitory potencies were similar to oxyphenylbutazone (IC50 = 1.9 microM) as a positive control.

Roles of active site aromatic residues in catalysis by ketosteroid isomerase from Pseudomonas putida biotype B.

The aromatic residues Phe-54, Phe-82, and Trp-116 in the hydrophobic substrate-binding pocket of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B have been characterized in their roles in steroid binding and catalysis. Kinetic and equilibrium binding analyses were carried out for the mutant enzymes with the substitutions Phe-54 --> Ala or Leu, Phe-82 --> Ala or Leu, and Trp-116 --> Ala, Phe, or Tyr. The removal of their bulky, aromatic side chains at any of these three positions results in reduced k(cat), particularly when Phe-82 or Trp-116 is replaced by Ala. The results are consistent with the binding interactions of the aromatic residues with the bound steroid contributing to catalysis. All the mutations except the F82A mutation increase K(m); the F82A mutation decreases K(m) by ca. 3-fold, suggesting a possibility that the phenyl ring at position 82 might be unfavorable for substrate binding. The K(D) values for d-equilenin, an intermediate analogue, suggest that a space-filling hydrophobic side chain at position 54, a phenyl ring at position 82, and a nonpolar aromatic or small side chain at position 116 might be favorable for binding the reaction intermediate. In contrast to the increased K(D) for equilenin, the enzymes with any substitutions at positions 54 and 116 display a decreased K(D) for 19-nortestosterone, a product analogue, indicating that Phe-54 and Trp-116 might be unfavorable for product binding. The crystal structure of F82A determined to 2.1-A resolution reveals that Phe-82 is important for maintaining the active site geometry. Taken together, our results demonstrate that Phe-54, Phe-82, and Trp-116 contribute differentially to the stabilization of steroid species including substrate, intermediate, and product.

Inhibitory effects of resveratrol analogs on unopsonized zymosan-induced oxygen radical production.

Resveratrol, a natural hydroxystilbene, has been reported to have anti-inflammatory and anticarcinogenic activities. Inhibitory effects of resveratrol and its analogs on reactive oxygen species (ROS) production in unopsonized zymosan-stimulated murine macrophage Raw264.7 cells, human monocytes, and neutrophils were analyzed to investigate if the anti-inflammatory and anticarcinogenic activities of resveratrol are related to the inhibition of ROS production. Resveratrol was a potent inhibitor of ROS production in both unopsonized zymosan-stimulated Raw264.7 cells and human monocytes and neutrophils. Resveratrol exhibited 50% inhibition values (IC50) of 17 microM in activated Raw264.7 cells, 18 microM in human monocytes, and 23 microM in human neutrophils. 3,5-Dihydroxy-4'-methoxystilbene or 3,4'-dimethoxy-5-hydroxystilbene exhibited IC50 values of 63 or 73 microM in Raw264.7 cells, 51 or >100 microM in human monocytes, and 10 or 37 microM in human neutrophils, respectively. Trimethylresveratrol, piceid, and 3,5-dihydroxy-4'-methoxystilbene-3-O-beta-D-glucoside were weak inhibitors of ROS production. Thus, resveratrol was identified as a potent inhibitor of ROS production, which might be one biochemical mechanism related to its anti-inflammatory and anticarcinogenic activities. The number and position of hydroxy substituents in resveratrol analogs seem to play an important role in the inhibitory potency of ROS production.

Hemistepsins with cytotoxic activity from Hemisteptia lyrata.

Two sesquiterpene lactones were isolated from Hemisteptia lyrata Bunge (Compositae). Their structures were elucidated by spectroscopic methods as recently known hemistepsin and the new 6-O-(2-methylpropenoyl)-3-hydroxy-4(15), 10(14), 11(13)-guaiatrien-12,8-olide. The two hemistepsin derivatives were examined for their in vitro cytotoxic activity against human cell lines such as UACC 62, HCT 15, UO-31, PC-3 and A549. Growth inhibitory property was determined by in vitro treatment of the respective cell lines using the sulforhodamine B assay (SRB). Both compounds exhibited significant cytotoxic activity (IC50 < 4.3 micrograms/ml) on all five human cell lines tested.

Effects of mutations in the C-terminal region of NIa protease on cis-cleavage between NIa and NIb.

Mutational analyses were carried out to investigate whether the nuclear inclusion protein a (NIa) C-terminal amino acids of turnip mosaic potyvirus play any roles in the cis-cleavage between NIa and NIb. The processing rate of the NIa-NIb junction sequence was decreased significantly by either V240D or Q243A mutation while little affected by F226D, V228E, K230E, I232D, or L235D mutation. The mutation of W212S, G213S, or I217D abolishing the cleavage at the NIb-CP or 6K1-cylindrical inclusion protein junction sequence decreased the processing rate to half the level of that of the wild type. Deletion of the C-terminal one (K230), two (S229 and K230), three (S229 to L231), or six amino acids (S229 to D234) as well as the insertion of five glycines between S229 and K230 or between S220 and Q221 did not affect significantly the cleavage while the deletion of 20 amino acids (Q218 to S237) decreased the processing rate to 73% of that of the wild type. These results rule out the possibility that the C-terminal region plays a role as a spacer in right placement of the NIa-NIb junction sequence and demonstrate that the C-terminal 20 amino acids from Q218 to S237 are not crucial for the cis-cleavage of the NIa-NIb junction sequence.

Structural analogues of cumambrin B from the flower of Chrysanthemum boreale.

The structural analogues of cumambrin B (1, 2, 3, 4) were isolated from the flower of Chrysanthemum boreale Makino. The structures of compounds were determined by two-dimensional 1H-1H COSY and 13C-1H COSY spectra with the aid of homonuclear and heteronuclear double resonance experiment. The stereochemistry of compounds has been verified from single crystal X-ray diffraction of cumambrin A (2). The antimicrobial activities of these guaianolides have been studied.