Structural and antifungal properties of a pathogenesis-related protein from wheat kernel

We have purified and characterized a protein from the water-soluble fraction of wheat kernel (Triticum aestivum cv. S. Pastore) consisting of a single polypeptide chain blocked at its N-terminus by a pyroglutamate residue; the complete amino acid sequence has been determined by automated sequence analysis performed on peptide fragments obtained by enzymatic hydrolyses of the protein. Homology studies have shown that this protein is very similar (97% sequence identity) to the previously characterized wheatwin1 as well as to other members of the pathogenesis-related (PR) proteins of class 4; in analogy with wheatwin1, we have termed this protein wheatwin2. Both wheatwin1 and wheatwin2 have specific antifungal activity toward the wide-host-range pathogenBotrytis cinerea and the wheat-specific pathogenic fungi of wheatFusarium culmorum andFusarium graminearum of groups 1 and 2. On the basis of their structural and functional properties, wheatwin1 and wheatwin2 can be classified as members of the PR4 protein family; this represents the first report concerning the presence of this kind of protein in wheat.     

Isolation and amino acid sequence of two new PR-4 proteins from wheat

We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2. Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of these residues were determined on the three-dimensional model of wheatwin1. Residue 88 was very accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.     

Recombinant Wheat Antifungal PR4 Proteins Expressed in Escherichia coli

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.     

Antifungal quinazolinones from marine-derived Bacillus cereus and their preparation

Two new quinazolinones alkaloids, R(+)-2-(heptan-3-yl)quinazolin-4(3H)-one (1) and (2R,3′R)+(2S,3′R)-2-(heptan-3-yl)-2,3-dihydroquinazolin-4(1H)-one (2) (a pair of epimers), as well as seven known analogues, 2-methylquinazolin-4(3H)-one (3), 2-benzylquinazolin-4(3H)-one (4), cyclo-(Pro-Ile), cyclo-(Pro-Leu), cyclo-(Pro-Val), cyclo-(Pro-Phe), and cyclo-(Tyr-Pro) were isolated from the n-butyl alcohol extract of the marine-derived bacterium Bacillus cereus 041381. The new compounds were identified by spectroscopic analysis and chemical synthesis. Four optical isomers 5−8 were also synthesized. Compounds 1−8 all showed moderate antifungal activity against Candida albicans with MIC values of 1.3−15.6 μM. Compound 5 exhibits the most powerful antifungal activity, which may reveal that S-configuration and 2,3-double bond were necessary for antifungal activity, and the racemization at C-2 and C-3′ reduced the antifungal activity.     

A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a ∼20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .The nucleotide sequence of AhPR10 reported in this paper is submitted to NCBI Nucleotide Sequence Database under the Accession number AY726607.     

Recombinant wheat antifungal PR4 proteins expressed in Escherichia coli.

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.     

Phytotoxic polyacetylenes from roots of Russian knapweed (Acroptilon repens (L.) DC.)

There are several factors thought to assist invasive weeds in colonization of ecosystems. One of these factors is allelopathy, the negative effect of chemicals produced by one plant on neighboring plants, frequently mediated through root exudates and other plant leachates. Acroptilon repens (Asteraceae) is one of the most invasive and ecologically threatening weed species in western North America. A bioassay-guided fractionation of the root extracts of this plant led to the isolation of five polyacetylenic compounds, of which one [5′-methoxy-1′-(5-prop-1-yn-1-yl-2-thienyl)-hexa-2′,4′-diyin-6′-yl acetate] was hitherto unknown. The structures of these compounds were elucidated on the basis of spectroscopic analysis (IR, ESIMS, ¹H, ¹³C NMR and 2D NMR). All of the compounds obtained, except 1-chloro-4-(5-penta-1,3-diyn-1-yl-2-thienyl)but-3-yn-2-ol, showed phytotoxic activity against Arabidopsis thaliana seedlings. The presence of 4′-chloro-1′-(5-penta-1,3-diyn-1-yl-2-thienyl)-but-2′-yn-3′-ol was detected in the root exudates of aeroponically grown A. repens plants. None of the polyacetylenes isolated in this study were found in Colorado soils collected between September 2006 and July 2007 in an A. repens colonized site. However, polyacetylene 5 in A. repens infested soil from Washington was found in June, 2007. Contrary to our previous report, the compound 7,8-benzoflavone (6) was not detected in root exudates, nor was it encountered in extracts of roots, aerial parts or infested soil. Since we could not repeat this work, the original report has been retracted [Stermitz, F.R., Bais, H.P., Foderaro, T.A., Vivanco, J.M., 2003. 7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed [A retraction]. Phytochemistry 64, 493-497.].     

Antifungal effect and mode of action of glochidioboside against Candida albicans membranes

Glochidioboside was obtained from Sambucus williamsii and its biological effect has not been reported. Its antifungal activity against pathogenic fungi and the mode of action involved in its effect were examined. Glochidioboside exerted antifungal effect with almost no hemolytic effect against human erythrocytes. To understand its antifungal mechanisms, membrane studies were done. Using two dyes, 3,3′-dipropylthiacarbocyanine iodide [DiSC₃(5)] and propidium iodide, membrane depolarization and permeabilization by glochidioboside were confirmed. Furthermore, the membrane-active mechanism was proven by synthesizing a model membrane, calcein-encapsulating large unilamellar vesicles (LUVs), and also by observing the influx of different sized fluorescent dyes, such as calcein, FD4 and FD10, into the fungal cells. The membrane-active action was pore-forming action with radii between 1.4 and 2.3 nm. Finally, three dimensional (3D) flow cytometric analysis showed the shrinkage of the fungal cells from the membrane damage. In conclusion, this study suggests that glochidioboside exerts an antifungal activity through a membrane-disruptive mechanism.     

A new Bauhinia monandra galactose-specific lectin purified in milligram quantities from secondary roots with antifungal and termiticidal activities

This work describes the purification in milligram quantities of a lectin from Bauhinia monandra secondary roots (BmoRoL) and its antifungal and termiticidal activities. The BmoRoL (6.2 mg) was isolated through ammonium sulfate fractionation and affinity chromatography on guar gel. Native lectin was resolved as a single band on polyacrylamide gel electrophoresis for basic proteins. Under denaturing and reducing conditions it appeared as a unique glycosylated polypeptide of 26 kDa. The highest agglutination activity of BmoRoL was found with glutaraldehyde-treated rabbit erythrocytes. BmoRoL showed antifungal activity against phytopathogenic species of Fusarium and was more active on Fusarium solani. The lectin also showed termiticidal activity on Nasutitermes corniger workers and soldiers with LC₅₀ of 0.09 and 0.395 mg ml⁻¹ for 12 days. In conclusion, BmoRoL is a new antifungal and termiticidal lectin that can be purified in milligram quantities and has potential biotechnological application for control of agricultural pests.     

Antifungal amides from Piper arboreum and Piper tuberculatum

In continuation of our study of the Piperaceae we have isolated several amides, mainly those bearing isobutyl, pyrrolidine, dihydropyridone and piperidine moieties. Bioactivity-guided fractionation of extracts from leaves of Piper arboreum yielded two new amides, N-[10-(13,14-methylenedioxyphenyl)-7(E),9(Z)-pentadienoyl]-pyrrolidine (1), arboreumine (2) together with the known compounds N-[10-(13,14-methylenedioxyphenyl)-7(E)-pentaenoyl]-pyrrolidine (3) and N-[10-(13,14-methylenedioxyphenyl)-7(E),9(E)-pentadienoyl]-pyrrolidine (4). Catalytic hydrogenation of 3 yielded the amide N-[10-(13,14-methylenedioxyphenyl)-pentanoyl]-pyrrolidine (5). We also have isolated six amides (6-11) and two antifungal cinnamoyl derivatives (12, 13) from seeds and leaves of Piper tuberculatum. Compounds 1-11 showed antifungal activity as determined by direct bioautography against Cladosporium sphaerospermum while compounds 3-4 and 6-13 also showed antifungal activity against C. cladosporioides.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Antimycobacterial,antibacterial and antifungal activities of Terminalia superba (Combretaceae)

The methanol extract from the stem bark of Terminalia superba (TSB), fractions (TSB1–7) and two compounds isolated following bio-assay guided fractionation namely 3,4′-di-O-methylellagic acid 3′-O-β-d-xylopyranoside (1) and 4′-O-galloy-3,3′-di-O-methylellagic acid 4-O-β-d-xylopyranoside (2) were evaluated for their antimycobacterial, antibacterial and antifungal activities. The broth microdilution, the microplate Alamar Blue assay (MABA) and the agar disc diffusion methods were used for the investigations. The results of the antimycobacterial assays showed that the crude extract, fractions TSB5–7 and compound 1 were able to prevent the growth of all the studied mycobacteria. The lowest minimal inhibitory concentration (MIC) value of 39.06 µg/ml for this extract was recorded on both M. smegmatis and M. tuberculosis MTCS2. The corresponding values were 19.53 µg/ml and 4.88 µg/ml for fractions and compounds respectively. The MIC determination results on other organisms indicated values ranging from 19.53 to 78.12 µg/ml for TSB and compound 2 on 90.9% of the tested organisms, meanwhile compound 1 as well as fractions TSB 6 and 7 exhibited detectable MIC values on all studied microorganisms. The overall results provide promising baseline information for the potential use of the crude extract from T. superba, fractions 6–7 and the tested compounds in the treatment of tuberculosis, bacterial and fungal infections.     

Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H-tetrazol-5-yl)methanamine derivatives

Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl₂ in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.     

Antibacterial phenolic components from Eriocaulon buergerianum

Five phenolic components, 1,3,6-trihydroxy-2,5,7-trimethoxyxanthone (1), 7,3′-dihydroxy-5,4′,5′-trimethoxyisoflavone (2), toralactone-9-O-β-d-glucopyranoside (3), patuletin-3-O-[2-O-E-feruloyl-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside] (4), patuletin-3-O-[β-d-glucopyranosyl-(1 → 3)-2-O-E-caffeoyl-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside] (5), along with 19 known compounds were isolated from Eriocaulon buergerianum (Eriocaulaceae). Their structures were determined by spectroscopic and chemical methods. All 24 isolated compounds were tested against the pathogenic bacteria Staphylococcus aureus (ATCC 25923); as a result, 10 compounds were found to exhibit antibacterial activity with MICs ranging from 32 to 256 μg/ml.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.     

Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1

We characterized a novel Bacillus thuringiensis isolate native to China (HS18-1) that shows a spherical crystal harboring two major proteins of about 70 and 130 kDa, and contains three novel cry genes (cry4Cb1, cry30Ga1, cry54-type). Furthermore, the cry4Cb1 and cry30Ga1 genes were expressed in Escherichia coli BL21 (DE3): pLysS. Insecticidal activity tests showed that the cry4Cb1 protein exhibited larvicidal activity against Aedes aegypti (Diptera) and the cry30Ga1 protein was toxic to both A. aegypti and P. xylostella (Lepidoptera).     

Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.     

Cytotoxic benzil and coumestan derivatives from Tephrosia calophylla

A benzil, calophione A, 1-(6′-Hydroxy-1′,3′-benzodioxol-5′-yl)-2-(6″-hydroxy-2″-isopropenyl-2″,3″-dihydro-benzofuran-5″-yl)-ethane-1,2-dione and three coumestan derivatives, tephcalostan B, C and D were isolated from the roots of Tephrosia calophylla. Their structures were deduced from spectroscopic data, including 2D NMR ¹H-¹H COSY and ¹³C-¹H COSY experiments. Compounds were evaluated for cytotoxicity against RAW (mouse macrophage cells) and HT-29 (colon cancer cells) cancer cell lines and antiprotozoal activity against various parasitic protozoa. Calophione A exhibited significant cytotoxicity with IC₅₀ of 5.00 (RAW) and 2.90 μM (HT-29), respectively.