Investigation of the antifungal activity and mechanism of action of LMWS-chitosan

Chitosan, a cationic polysaccharide, has been widely used as a dietary supplement and in a variety of pharmacological and biomedical applications. The antifungal activity and mechanism of action of low molecular weight water-soluble chitosan (LMWS-chitosan) were studied in fungal cells and vesicles containing various compositions of fungal lipids. LMWS-chitosan showed strong antifungal activity against various pathogenic yeasts and hyphae-forming fungi but no hemolytic activity or cytotoxicity against mammalian cells. The degree of calcein leakage was assessed on the basis of lipid composition (PC/CH; 10:1, w/w). Our result showing that LMWS-chitosan interacts with liposomes demonstrated that chitosan induces leakage from zwitterionic lipid vesicles. Confocal microscopy revealed that LMWS-chitosan was located in the plasma membrane. Finally, scanning electron microscopy revealed that LMWS-chitosan causes significant morphological changes on fungal surfaces. Its potent antibiotic activity suggests that LMWS-chitosan is an excellent candidate as a lead compound for the development of novel anti-infective agents.     

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

HP (2-20) derived from the amino terminal region of helicobacterpylori ribosomal protein L1 exerts its antifungal effects by damaging the plasma membranes of Candida albicans.

The fungicidal effects of the peptide HP (2-20). derived from the N-terminal sequence of Helicobacter pylori ribosomal protein L1 (RPL1). have been investigated. HP (2-20) displays a strong fungicidal activity against various fungi, without haemolytic activity against human erythrocyte cells, and the fungicidal activity is inhibited by Ca2+ and Mg2+ ions. In order to investigate the fungicidal mechanism(s) of HP (2-20). the amount of intracellular trehalose was measured in C. albicans. It was found that the amounts of intracellular trehalose were decreased when HP (2-20) was used. The action of the peptide against fungal cell membranes was further examined by the potassium-release test; HP (2-20) was found to increase the amount of K+ released from the cells. Furthermore, HP (2-20) caused significant morphological changes, as shown by scanning electron microscopy, and by testing the membrane disrupting activity using liposomes (phosphatidyl choline/cholesterol; 10: 1, w/w). Our results suggest that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membranes, via pore formation or direct interaction with the lipid bilayers.     

Fungicidal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Candida albicans

The antifungal activity and mechanism of a 23-mer peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed strong antifungal activity against yeast and mold. To investigate the antifungal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. Candida albicans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide(PI) staining, which was similar to that of Melittin than untreated cells. Confocal microscopy showed that the peptide was located in the plasma membrane. The action of peptides against fungal cell membranes was examined by treating prepared protoplasts of C. albicans with the peptide and lipid vesicle titration test. The result showed that the peptide prevented the regeneration of fungal cell walls and induced release of the fluorescent dye trapped in the artificial membrane vesicles, indicating that the peptide exerts its antifungal activity by acting on the plasma lipid membrane.     

Restoration of heat shock protein70 suppresses gastric mucosal inducible nitric oxide synthase expression induced by Helicobacter pylori

Heat shock proteins (HSPs) are crucial for the maintenance of cell integrity during normal cellular growth as well as during pathophysiological conditions. While functioning mainly as molecular chaperones, HSPs also appear to be involved in diverse biological activities, such as apoptosis, carcinogenesis, and cytoprotection from cytotoxic damage. Infection with Helicobacter pylori causes inflammation in the gastric mucosa, leading to gastritis, gastric ulcers, duodenal ulcer disease, and even gastric cancer, but the role of HSPs in H. pylori-associated gastropathy is not known. Using two-dimensional electrophoretic analysis, we have observed significant shifts in HSP profiles after H. pylori infection in RGM-1 cells. We therefore evaluated the effect of treatments that induce HSPs on H. pylori-induced inducible nitric oxide synthase (iNOS) expression. We found that H. pylori infection significantly attenuated the expression of HSP70, whereas exposure of cells to noncytotoxic heat shock or geranylgeranylacetone restored HSP70 expression, as well as suppressing the expression of iNOS, a major cause of H. pylori-induced gastric tissue damage. Our results suggest that induction of HSP70 confers cytoprotection against H. pylori infection by inhibiting the expression of iNOS. In conclusion, these results provide important insights into the flux in HSPs profiles in response to H. pylori infection and highlight the cytoprotective role of HSP70 in H. pylori infection.     

Structural and Functional Analysis of BipA,a Regulator of Virulence in Enteropathogenic Escherichia coli

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3′, 5′-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes to small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.     

Processing of Ada protein by two serine endoproteases Do and So from Escherichia coli

Two soluble serine proteases Do and So from Escherichia coli were found to distinctively cleave the purified, 39 kDa Ada protein into fragments with sizes of 12-31 kDa. Protease So appears to generate a C-terminal 19 kDa polypeptide, similarly to OmpT protease. In addition, the purified 19 kDa C-terminal half of Ada protein can be further processed mainly to an 18 kDa fragment by protease So and to a 12 kDa by protease Do. These results suggest that proteases Do and So are involved in endogenous cleavage of Ada protein, which may play a role in down-regulating the adaptive response to alkylating agents.     

Arsenic trioxide represses constitutive activation of NF-kappaB and COX-2 expression in human acute myeloid leukemia, HL-60

It has been proposed that eukaryotic nuclear factor nuclear factor kappa-B (NF-kappaB) and cyclooxygenase-2 (COX-2) are implicated in the pathogenesis of many human diseases including cancer. Arsenic has been widely used in medicine in Oriental countries. Recent studies have shown that arsenic trioxide (As(2)O(3)) could induce in vitro growth inhibition and apoptosis of malignant lymphocytes, and myeloma cells. However, the molecular mechanisms by which As(2)O(3) initiates cellular signaling toward cell death are still unclear. In the present study, the effects of As(2)O(3) on NF-kappaB and COX-2 expression in HL-60 cells were investigated. As(2)O(3) suppressed DNA-binding activity of NF-kappaB composed of p65/p50 heterodimer through preventing the degradation of IkappaB-alpha and the nuclear translocation of p65 subsequently as well as interrupting the binding of NF-kappaB with their consensus sequences. Inhibitory effect of As(2)O(3) on NF-kappaB DNA activity was dependent upon intracellular glutathione (GSH) and H(2)O(2) level, but not superoxide anion. Futhermore, we found that As(2)O(3) also downregulated the expression of COX-2, which has NF-kappaB binding site on its promoter through repressing the NF-kappaB DNA-binding activity.