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1.
Figure 7

Figure 7. Structures of FtsZ inhibitors and scaffolds.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

General scaffolds for small molecule library compounds (A). Structure of berberine (B). FtsZ inhibitors identified from the initial high-throughput screen (C). FtsZ inhibitors identified from SAR studies (D).

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.
2.
Figure 1

Figure 1. Wolbachia ftsZ gene expression in various developmental stages of B. malayi.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

Female adult worm, male adult worm, microfilaria, L3 and L4 were analyzed. The ratio of ftsZ to 16S rRNA (A) represents ftsZ gene expression, while the ratio of Wolbachia 16S to B. malayi 18S rRNA (B) represents the relative abundance of Wolbachia in B. malayi. The data obtained from triplicate samples are expressed as a mean ± standard deviation.

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.
3.
Figure 2

Figure 2. Expression and purification of recombinant FtsZ proteins.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

FtsZ protein from Wolbachia (A) and E. coli (B) were expressed in E. coli with a His-tag at the N-terminus and purified by nickel-affinity chromatography. The apparent molecular weight (indicated) from SDS–PAGE was consistent with the predicted molecular size. Protein marker (M), total protein lysate (T), insoluble proteins (In), soluble (S) and purified FtsZ proteins (P) are shown.

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.
4.
Figure 4

Figure 4. Berberine sulfate inhibition of the GTPase activity.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

Enzyme activity of wBm-FtsZ (•) and Ec-FtsZ (□) was determined in the presence of 0, 25, 50, 100, 200, 400, 600, 800, and 1000 µM berberine sulfate. The data obtained from triplicate samples are expressed as a mean ± standard deviation.

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.
5.
Figure 8

Figure 8. Inhibition of GTPase activity by small molecules.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

wBm-FtsZ (▪ and •) and Ec-FtsZ (□ and ○) were compared. Panel A, compounds were tested at the concentration of 30, 40, 50, 60, 70, 80, 90, and 100 µM and the experiment were performed in duplicate, the mean value was plotted. Panel B, compounds were tested at the concentration of 10, 20, 40, 60, 80, and 100 µM and the experiments were performed in triplicate, the mean ± standard deviation was plotted.

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.
6.
Figure 3

Figure 3. GTPase activity of recombinant wBm-FtsZ.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

Panel A, activity was determined indirectly by measuring a decrease in NADH concentration by its absorbance at 340 nm. FtsZ hydrolyzes GTP into GDP and inorganic phosphate. The GDP product is used as a substrate by pyruvate kinase (PK) in the presence of phosphoenol pyruvate (PEP) to yield GTP and pyruvic acid as products. Pyruvic acid is used as a substrate in the presence of NADH by lactate dehydrogenase (LDH) to generate lactate and NAD. The consumption of NADH is proportional to GTPase activity. Panel B, comparison of the GTPase activity of wBm-FtsZ and Ec-FtsZ. A control without enzyme was also included. Activity was indicated by a decrease of NADH measured by absorbance at 340 nm.

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.
7.
Figure 5

Figure 5. Effect of berberine sulfate on B. malayi parasites in culture.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

Motility of adult female (A) and male (D) worms, and microfilariae (C) was examined following 6 days exposure to varying amounts (5–40 µM) of berberine sulfate. 10 µM doxycycline was included as a control. Motility was scored as described [38] and expressed as % of motility relative to motility scored on day 0 of the experiment. Micofilariae production (B) was determined at each time point by counting the number of microfilaria present in 1 mL spent culture media. The data obtained from triplicate samples are expressed as a mean ± standard deviation.

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.
8.
Figure 6

Figure 6. Berberine sulfate inhibition of E. coli growth.. From: Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy.

Panel A, overnight growth of E. coli was determined in the presence of various concentrations of berberine sulfate. Percentage of growth is indicated as 100×(OD600 nm with berberine/OD600 nm without berberine). The data obtained from triplicate samples are expressed as a mean ± standard deviation. Panel B, log-phase (5 h) growth (OD600 nm) of E. coli was determined in the presence of various concentrations (10–70 µM) of berberine sulphate. Panel C, DIC micrographs of E. coli untreated (0 µM) or treated with 40 µM or 80 µM berberine sulfate. Panel D, effect of berberine sulfate on E. coli viability. Viability of berberine sulfate-treated (24 hours) cells was evaluated by plating serial dilutions (10−2–10−7) of bacteria (Output shown in duplicate) on a petri dish and incubation overnight at 30 °C. The number of bacteria present in the inoculum used in the experiment (Input) is also shown.

Zhiru Li, et al. PLoS Negl Trop Dis. 2011 November;5(11):e1411.

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