Antibiotic activity and synergistic effect of antimicrobial peptide against pathogens from a patient with gallstones

HP (2-20) is a peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1 that has been shown to have antimicrobial activity against various species of bacteria. When we tested the effects of HP (2-20), we found that this peptide displayed strong activity against pathogens from a patient with gallstones, but it did not have hemolytic activity against human erythrocytes. We also found that HP (2-20) had potent activity against cefazolin sodium-resistant bacterial cell lines, and that HP (2-20) and cefazolin sodium had synergistic effects against cell lines resistant to the latter. To investigate the mechanism of action of HP (2-20), we performed fluorescence activated flow cytometry using pathogens from the patient with gallstones. As determined by propidium iodide (PI) staining, pathogenic bacteria treated with HP (2-20) showed higher fluorescence intensity than untreated cells, similar to melittin-treated cells, and that HP (2-20) acted in an energy- and salt-dependent manner. Scanning electron microscopy showed that HP (2-20) caused significant morphological alterations in the cell surface of pathogens from the patient with gallstones. By determining their 16S rDNA sequences, we found that both the pathogens from the patient with gallstones and the cefazolin sodium-resistant cell lines showed 100% homology with sequences from Pseudomonas aeruginosa. Taken together, these results suggest that HP (2-20) has antibiotic activity and that it may be used as a lead drug for the treatment of acquired pathogens from patients with gallstones and antibiotic-resistant cell lines.     

Antibacterial synergism of novel antibiotic peptides with chloramphenicol

HP (2-20) is an antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously tested whether several analogues of HP (2-20), with amino acid substitutions that increased or decreased net hydrophobicity, could be useful as therapeutic agents. In the present study, we show that substituting Gln and Asp for Trp at positions 17 and 19, respectively, of HP (2-20) (peptide A3) had potent antibacterial activity in minimal inhibition concentration and minimal bactericidal concentration without having hemolytic activity. In contrast, when we decreased hydrophobicity by substituting Leu or Phe for Ser at positions 12 and 19, respectively, of HP (2-20) (Anal 4, Anal 5), there was no significant effect on antibacterial activity. We found that A3 acted synergistically with chloramphenicol against bacterial cells. Fluorescence activated flow cytometry showed that A3-treated cells had higher fluorescence intensity than untreated cells, similar to that of melittin-treated cells. Furthermore, A3 caused significant morphological alterations of Staphylococcus aureus and Pseudomonas aeruginosa, as shown by scanning electron microscopy. Our results suggest that peptide A3 may be useful for the design of novel antibiotic peptides that possess high bacterial cell selectively and synergistic effects with conventional antibiotic agents but lack hemolytic activity.     

Metallothionein-III prevents gamma-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.     

Selective cytotoxicity following Arg-to-Lys substitution in tritrpticin adopting a unique amphipathic turn structure

In antimicrobial peptides, the cationic property due to basic amino acids has been widely recognized as an important factor to promote electrostatic interaction with negatively charged phospholipids. However, little is known about the differences between two basic residues, Arg and Lys, in membrane binding affinity. Tritrpticin is an Arg- or Trp-rich antimicrobial peptide with a broad spectrum of antibacterial and antifungal activity. To investigate the structural and functional differences between Arg and Lys residues, here we designed and synthesized Arg-containing peptides, tritrpticin and SYM11, and their counterpart Lys-substituted peptides, TRK and SYM11KK, respectively. Although there were no remarkable conformational differences between Arg-containing and Lys-substituted peptides, TRK and SYM11KK exhibited almost two-fold enhanced antibacterial activity but significantly reduced hemolytic activity as compared to tritrpticin and SYM11, respectively. Furthermore, Arg-containing peptides showed strong binding affinity to both zwitterionic and anionic liposomes, whereas Lys-substituted peptides interacted weakly with zwitterionic liposomes but strongly with anionic liposomes. These results suggest that the primary amine of Lys interacts less electrostatically with zwitterionic phospholipids than the guanidinium group of Arg. Our results obtained in this study may be helpful in the design of drugs that target negatively charged phospholipids.     

L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway

l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.Abbreviations NAC N-acetyl-l-cysteine - ROS reactive oxygen species - _m mitochondrial membrane potentialJae Seung Kang and Daeho Cho contributed equally to this work.     

Measles virus infects and suppresses proliferation of T lymphocytes from transgenic mice bearing human signaling lymphocytic activation molecule

Humans are the only natural reservoir of measles virus (MV), one of the most contagious viruses known. MV infection and the profound immunosuppression it causes are currently responsible for nearly one million deaths annually. Human signaling lymphocytic activation molecule (hSLAM) was identified as a receptor for wild-type MV as well as for MV strains prepared as vaccines. To better evaluate the role of hSLAM in MV pathogenesis and MV-induced immunosuppression, we created transgenic (tg) mice that expressed the hSLAM molecule under the control of the lck proximal promoter. hSLAM was expressed on CD4(+) and CD8(+) T cells in the blood and spleen and also on CD4(+), CD8(+), CD4(+) CD8(+), and CD4(-) CD8(-) thymocytes. Wild-type MV, after limited passage on B95-8 marmoset B cells, and the Edmonston laboratory strain of MV infected hSLAM-expressing cells. There was a direct correlation between the amount of hSLAM expressed on the cells' surface and the degree of viral infection. Additionally, MV infection induced downregulation of receptor hSLAM and inhibited cell division and proliferation of hSLAM(+) but not hSLAM(-) T cells. Therefore, these tg mice provide the opportunity for analyzing and comparing MV-T cell interactions and MV pathogenesis in cells expressing only the hSLAM MV receptor with those of tg mice whose T cells selectively express another MV receptor, CD46.     

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella tryphimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect but that the alpha-helical property is not connected with the enhanced antibiotic activity.     

Comparative analysis of nuclear proteins of B cells in different developmental stages

The developmental stage-specific regulation of V(D)J recombination, a gene rearrangement process of antigen receptor gene segments, is tightly controlled in cells. Here we screened proteins uniquely or differentially expressed among three developmentally distinguishable B cells (pro-B, pre-B and mature B cells) using two-dimensional gel electrophoresis and mass spectrometry. Chromatin assembly factor 1 was uniquely expressed in pro-B cells. Purine nucleotide phosphorylase, LCK, E2A and many other unidentified proteins were dominantly present in the nucleus at the early stage of B cell development where the V(D)J recombination process occurs. Also, few proteins including guanidine nucleotide binding proteins were exclusively expressed in pre-B cell. Such findings can provide some information to help understand the developmental regulation of gene rearrangement occurring during B cell development.     

Regulation of Staphylococcus epidermidis-induced IFN-gamma in whole human blood: the role of endogenous IL-18, IL-12, IL-1, and TNF

Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-gamma) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-gamma synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-gamma and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-gamma and IL-1beta levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-gamma (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-gamma production. Blocking endogenous IL-12 and TNF reduced IFN-gamma production by 69 and 36%. S. epidermidis-induced TNF-alpha was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-gamma which is IL-18, IL-12 and TNF-dependent, but IL-1 independent.     

Differential gene regulation by specific gain-of-function JNK1 proteins expressed in Swiss 3T3 fibroblasts

The c-Jun N-terminal kinases (JNKs) are encoded by three genes that yield 10 isoforms through alternative mRNA splicing. The roles of each JNK isoform in the many putative biological responses where the JNK pathway is activated are still unclear. To examine the cellular responses mediated by different JNK isoforms, gain-of-function JNK1 polypeptides were generated by fusing the upstream mitogen-activated protein kinase kinase, MKK7, with p46JNK1alpha or p46JNK1beta. The MKK7-JNK fusion proteins, which exhibited constitutive activity in 293T cells, were stably expressed in Swiss 3T3 fibroblasts using retrovirus-mediated gene transfer. Swiss 3T3 cells expressing either of the MKK7-JNK polypeptides were equally sensitized to induction of cell death following serum withdrawal. To search for other cellular responses that may be selectively regulated by the JNK1 isoforms, the gene expression profiles of Swiss 3T3 cells expressing MKK7-JNK1alpha or MKK7-JNK1beta were compared with empty vector-transfected control cells. Affymetrix Genechips identified 46 genes for which expression was increased in MKK7-JNK-expressing cells relative to vector control cells. Twenty genes including those for c-Jun, MKP-7, interluekin-1 receptor family member ST2L/ST2, and c-Jun-binding protein were induced similarly by MKK7-JNK1alpha and MKK7-JNK1beta proteins, whereas 13 genes were selectively increased by MKK7-JNK1alpha and 13 genes were selectively increased by MKK7-JNK1beta. The set of genes selectively induced by MKK7-JNK1beta included a number of known interferon-stimulated genes (ISG12, ISG15, IGTP, and GTPI). Consistent with these gene expression changes, Swiss 3T3 cells expressing MKK7-JNK1beta exhibited increased resistance to vesicular stomatitis virus-induced cell death. These findings reveal evidence for JNK isoform-selective gene regulation and support a role for distinct JNK isoforms in specific cellular responses.