Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon1

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.

Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides.

Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.

Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 5′-methoxyhydnocarpin, a multidrug pump inhibitor

Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal plants producing berberine were found also to synthesize an inhibitor of the NorA MDR pump of a human pathogen Staphylococcus aureus. The inhibitor was identified as 5′-methoxyhydnocarpin (5′-MHC), previously reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy. 5′-MHC is an amphipathic weak acid and is distinctly different from the cationic substrates of NorA. 5′-MHC had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely inhibited by 5′-MHC. The level of accumulation of berberine in the cells was increased strongly in the presence of 5′-MHC, indicating that this plant compound effectively disabled the bacterial resistance mechanism against the berberine antimicrobial.

Structure–activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II

Buforin II is a 21-aa potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 1–4), an extended helical region (residues 5–10), a hinge (residue 11), and a C-terminal regular α-helical region (residues 12–21). To elucidate the structural features of buforin II that are required for its potent antimicrobial activity, we synthesized a series of N- and C-terminally truncated or amino acid-substituted synthetic buforin II analogs and examined their antimicrobial activity and mechanism of action. Deletion of the N-terminal random coil region increased the antibacterial activity ≈2-fold, but further N-terminal truncation yielded peptide analogs with progressively decreasing activity. Removal of four amino acids from the C-terminal end of buforin II resulted in a complete loss of antimicrobial activity. The substitution of leucine for the proline hinge decreased significantly the antimicrobial activity. Confocal fluorescence microscopic studies showed that buforin II analogs with a proline hinge penetrated the cell membrane without permeabilization and accumulated in the cytoplasm. However, removal of the proline hinge abrogated the ability of the peptide to enter cells, and buforin II analogs without a proline hinge localized on the cell surface, permeabilizing the cell membrane. In addition, the cell-penetrating efficiency of buforin II and its truncated analogs, which depended on the α-helical content of the peptides, correlated linearly with their antimicrobial potency. Our results demonstrate clearly that the proline hinge is responsible for the cell-penetrating ability of buforin II, and the cell-penetrating efficiency determines the antimicrobial potency of the peptide.

Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia

Ischemic stroke is the most common life-threatening neurological disease and has limited therapeutic options. One component of ischemic neuronal death is inflammation. Here we show that doxycycline and minocycline, which are broad-spectrum antibiotics and have antiinflammatory effects independent of their antimicrobial activity, protect hippocampal neurons against global ischemia in gerbils. Minocycline increased the survival of CA1 pyramidal neurons from 10.5% to 77% when the treatment was started 12 h before ischemia and to 71% when the treatment was started 30 min after ischemia. The survival with corresponding pre- and posttreatment with doxycycline was 57% and 47%, respectively. Minocycline prevented completely the ischemia-induced activation of microglia and the appearance of NADPH-diaphorase reactive cells, but did not affect induction of glial acidic fibrillary protein, a marker of astrogliosis. Minocycline treatment for 4 days resulted in a 70% reduction in mRNA induction of interleukin-1β-converting enzyme, a caspase that is induced in microglia after ischemia. Likewise, expression of inducible nitric oxide synthase mRNA was attenuated by 30% in minocycline-treated animals. Our results suggest that lipid-soluble tetracyclines, doxycycline and minocycline, inhibit inflammation and are neuroprotective against ischemic stroke, even when administered after the insult. Tetracycline derivatives may have a potential use also as antiischemic compounds in humans.

Are amoxycillin and folate inhibitors as effective as other antibiotics for acute sinusitis? A meta-analysis

Objectives: To examine whether antibiotics are indicated in treating uncomplicated acute sinusitis and, if so, whether newer and more expensive antibiotics with broad spectra of antimicrobial activity are more effective than amoxycillin or folate inhibitors.

Characterization of Two Novel Type I Ribosome-Inactivating Proteins from the Storage Roots of the Andean Crop Mirabilis expansa1

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.

Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2−/− mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2−/− mice, we used suppressive subtractive hybridization between livers from Usf2−/− and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2−/− hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox.

Reactive oxygen intermediates generated by the phagocyte NADPH oxidase are critically important components of host defense. However, these highly toxic oxidants can cause significant tissue injury during inflammation; thus, it is essential that their generation and inactivation are tightly regulated. We show here that an endogenous proline-arginine (PR)-rich antibacterial peptide, PR-39, inhibits NADPH oxidase activity by blocking assembly of this enzyme through interactions with Src homology 3 domains of a cytosolic component. This neutrophil-derived peptide inhibited oxygen-dependent microbicidal activity of neutrophils in whole cells and in a cell-free assay of NADPH oxidase. Both oxidase inhibitory and direct antimicrobial activities were defined within the amino-terminal 26 residues of PR-39. Oxidase inhibition was attributed to binding of PR-39 to the p47phox cytosolic oxidase component. Its effects involve both a polybasic amino-terminal segment and a proline-rich core region of PR-39 that binds to the p47phox Src homology 3 domains and, thereby, inhibits interaction with the small subunit of cytochrome b558, p22phox. These findings suggest that PR-39, which has been shown to be involved in tissue repair processes, is a multifunctional peptide that can regulate NADPH oxidase production of superoxide anion O2-. thus limiting excessive tissue damage during inflammation.