Type 2 human papillomavirus E7 attenuates E-cadherin expression in human keratinocytes.

Human papillomaviruses (HPVs) are known to utilize the down-regulation of epithelial (E)-cadherin, a major component of adherens junctions of keratinocytes, to evade host immune surveillance in high-risk group. However, the effects of HPV on the function of E-cadherin in low-risk groups remain unknown. We investigated whether type 2 HPV (HPV-2) E7 could induce alterations in E-cadherin expression in transiently transfected keratinocytes and cell lines expressing HPV-2 E7. To examine the expression pattern of E-cadherin in cutaneous warts and normal skin samples, immunohistochemical analysis was performed. Quantitative real-time polymerase chain reactions, luciferase assays, western blot, immunocytochemistry, and electron microscopy were used to evaluate the mRNA and protein expression levels of E-cadherin in normal human epidermal keratinocytes transfected with HPV-2 E7 plasmid DNA or E7-specific siRNA and in E7-expressing cell lines. E-cadherin expression levels in HPV-2 positive cutaneous warts were significantly decreased compared to those in normal skin (p < 0.05). Similarly, the mRNA and protein expression levels of E-cadherin in E7 transiently transfected cells were significantly decreased compared to those in empty vector-transfected cells. The decreases were restored by transfection with E7-specific siRNA (p < 0.05). Likewise, cell lines expressing E7 showed a decreased expression of E-cadherin. When the cells were cultured in low attachment plates, cell-to-cell aggregation was inhibited. Taken together, our data suggest that HPV-2 E7, the causative agent of cutaneous warts, could mediate the transcriptional repression of E-cadherin.

Epigallocatechin-3-gallate Can Prevent Type 2 Human Papillomavirus E7 from Suppressing Interferon-Stimulated Genes.

Human papillomavirus (HPV) in high-risk groups is known to suppress the type I interferon (IFN) signaling pathway leading to the transcription of interferon-stimulated genes (ISGs), which have many antiviral functions. However, the effects of HPV on the action of various ISGs in low-risk groups are not fully understood. We aimed to investigate whether antiviral ISGs are expressed in transfected keratinocytes with type 2 HPV (HPV-2) E7. The mRNA and protein expressions of ISGs and type I IFN signaling pathway components were evaluated by quantitative real-time polymerase chain reaction, western blot, immunofluorescence, and/or immunohistochemistry. Compared with normal skin, mRNA expression of all ISGs in HPV-2 positive cutaneous warts was significantly decreased (p < 0.05). In comparison with empty vector transfection, E7 transfection significantly down-regulated the mRNA and protein expressions of ISGs and type I IFN signaling pathway components, which were significantly up-regulated by E7 siRNA transfection (p < 0.05). Interestingly, epigallocatechin-3-gallate (EGCG) pretreatment up-regulated the mRNA and protein expressions of ISGs and type I IFN signaling pathway components, which were significantly down-regulated by E7 transfection (p < 0.05). Our results demonstrate that EGCG is a potential candidate for cutaneous wart prevention.

2,3,22,23-tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene, an acyclic triterpenoid isolated from the seeds of Alpinia katsumadai, Inhibits acyl-CoA : cholesterol acyltransferase activity.

In order to isolate a cholesterol-lowering compound from Alpinia katsumadai, an inhibitor for acyl-CoA : cholesterol acyltransferase (ACAT), an enzyme responsible for the cholesterol ester formation in liver, was purified, its chemical structure was determined, and in vivo and in vitro inhibition activities were performed. In a high fat diet mouse model, we discovered that the ethanol extract of Alpinia katsumadai reduced plasma cholesterol, triglyceride, and low density lipoprotein (LDL) levels. An acyclic triterpenoid showing ACAT inhibitory activity was isolated from the extract of seeds of A. katsumadai. By NMR spectroscopic analysis of its (1)H-NMR, (13)C-NMR, (1)H-(1)H correlation spectroscopy, heteronuclear multiple bond connectivity (HMBC), hetero multiquantum coherence (HMQC) and nuclear Overhauser effect, chemical structure of 2,3,22,23-tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (1), were elucidated. The acyclic triterpenoid was found to be responsible for the ACAT inhibition activities of rat liver microsomes with IC(50) values of 47.9 µM. It also decreased cholesteryl ester formation with IC(50) values of 26 µM in human hepatocyte HepG2 cell. The experimental study revealed that the ethanol extract of A. katsumadai has a hypolipemic effect in high fat diet mice, and the isolated acyclic triterpenoid has ACAT inhibition activity, showing a potential novel therapeutic approach for the treatment of hyperlipidemia and atherosclerosis.

The conformation and CETP inhibitory activity of [10]-dehydrogingerdione isolated from Zingiber officinale.

In the course of searching for cholesteryl ester transfer protein (CETP) inhibitors from natural sources, a new type of CETP inhibitor, [10]-dehydrogingerdione (1), was isolated from the extract of rhizomes of Zingiber officinale Roscoe. By NMR spectroscopic analysis of its (1)HNMR, (13)C-NMR, and (1)H-(1)H COSY, HMBC, HMQC and NOESY, more precise structure, compared with its originally proposed structures, of [10]-dehydrogingerdione has been elucidated. This active compound inhibited human plasma CETP with IC(50) values of 35 µM.

Antimicrobial and anti-inflammatory effects of Cecropin A(1-8)-Magainin2(1-12) hybrid peptide analog p5 against Malassezia furfur infection in human keratinocytes.

The lipophilic fungus Malassezia furfur (M. furfur) is a commensal microbe associated with several chronic diseases such as pityriasis versicolor, folliculitis, and seborrheic dermatitis. Because M. furfur-related diseases are difficult to treat and require prolonged use of medications, the treatment for M. furfur-related skin diseases is supposed to gain control over M. furfur growth and the inflammation associated with it, as well as to prevent secondary infections. In this study, we investigated the antifungal and anti-inflammatory effects of cecropin A(1-8)-magainin 2(1-12) hybrid peptide analog P5 on M. furfur. The minimal inhibitory concentration of P5 against M. furfur was 0.39 µM, making it 3-4 times more potent than commonly used antifungal agents such as ketoconazole (1.5 µM) or itraconazole (1.14 µM). P5 efficiently inhibited the expression of IL-8 and Toll-like receptor 2 in M. furfur-infected human keratinocytes without eukaryotic cytotoxicity at its fungicidal concentration. Moreover, P5 significantly downregulated NF-κB activation and intracellular calcium fluctuation, which are closely related with enhanced responses of keratinocyte inflammation induced by M. furfur infection. Taken together, these observations suggest P5 may be a potential therapeutic agent for M. furfur-associated human skin diseases because of its distinct antifungal and anti-inflammatory action.

Insect ornithine decarboxylase (ODC) complements SPE1 knock-out of yeast Saccharomyces cerevisiae.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth, differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. Mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%, respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of > 4% of the wild-type strain in polyaminefree media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore, its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system.

Enhanced calreticulin expression promotes calcium-dependent apoptosis in postnatal cardiomyocytes.

Calreticulin (CRT) is one of the major Ca2+ binding chaperone proteins of the endoplasmic reticulum (ER) and an unusual luminal ER protein. Postnatally elevated expression of CRT leads to impaired development of the cardiac conductive system and may be responsible for the pathology of complete heart block. In this study, the molecular mechanisms that affect Ca2+-dependent signal cascades were investigated using CRT-overexpressing cardiomyocytes. In particular, we asked whether calreticulin plays a critical role in the activation of Ca2+-dependent apoptosis. In the cells overexpressing CRT, the intracellular calcium concentration was significantly increased and the activity of PKC and level of SECAR2a mRNA were reduced. Phosphorylation of Akt and ERKs decreased compared to control. In addition the activity of the anti-apoptotic factor, Bcl-2, was decreased and the activities of pro-apoptotic factor, Bax, p53 and caspase 8 were increased, leading to a dramatic augmentation of caspase 3 activity. Our results suggest that enhanced CRT expression in mature cardiomyocytes disrupts intracellular calcium regulation, leading to calcium-dependent apoptosis.

The global regulator Spx functions in the control of organosulfur metabolism in Bacillus subtilis.

Spx is a global transcriptional regulator of the oxidative stress response in Bacillus subtilis. Its target is RNA polymerase, where it contacts the alpha subunit C-terminal domain. Recently, evidence was presented that Spx participates in sulfate-dependent control of organosulfur utilization operons, including the ytmI, yxeI, ssu, and yrrT operons. The yrrT operon includes the genes that function in cysteine synthesis from S-adenosylmethionine through intermediates S-adenosylhomocysteine, ribosylhomocysteine, homocysteine, and cystathionine. These operons are also negatively controlled by CymR, the repressor of cysteine biosynthesis operons. All of the operons are repressed in media containing cysteine or sulfate but are derepressed in medium containing the alternative sulfur source, methionine. Spx was found to negatively control the expression of these operons in sulfate medium, in part, by stimulating the expression of the cymR gene. In addition, microarray analysis, monitoring of yrrT-lacZ fusion expression, and in vitro transcription studies indicate that Spx directly activates yrrT operon expression during growth in medium containing methionine as sole sulfur source. These experiments have uncovered additional roles for Spx in the control of gene expression during unperturbed, steady-state growth.

Mutational analysis of the Bacillus subtilis RNA polymerase alpha C-terminal domain supports the interference model of Spx-dependent repression.

The Spx protein of Bacillus subtilis exerts both positive and negative transcriptional control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (alphaCTD). Thus, transcription of the srf operon at the onset of competence development, which requires the ComA response regulator of the ComPA signal transduction system, is repressed by Spx-alphaCTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the alpha1 region of alphaCTD. Alanine-scanning mutagenesis of B. subtilis alphaCTD uncovered residue positions required for Spx function and ComA-dependent srf transcriptional activation. Analysis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicate that Spx interferes with ComA-alphaCTD interaction and that residues Y263, C265, and K267 of the alpha1 region lie within overlapping ComA- and Spx-binding sites for alphaCTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.

CuiD is a crucial gene for survival at high copper environment in Salmonella enterica serovar Typhimurium.

Copper ion is an essential micronutrient but it is also extremely cytotoxic when it exists in excess. Our studies have shown that Salmonella enterica serovar Typhimurium can survive potentially lethal copper exposures by the way of copper efflux system. A copper ion inducible gene was identified in virulent S. typhimurium by using the technique of MudJ (Km, lac)-directed lacZYA operon fusions. A copper ion inducible strain LF153 (cuiD::MudJ) has been identified. The cuiD mutant exhibits a copper sensitive phenotype but possesses normal resistance to other metal ions, and lost DMP oxidase activity. Therefore, we suggest that cuiD is an important gene for copper homeostasis and the copper resistance response. The copper sensitive phenotype was complemented by pYL3.0 carrying cuiD+. Sequence analysis showed cuiD contains 1,614 bp encoding a 536 amino acid with a 27 amino acid signal peptide and a 509 amino acid residues comprising the mature peptide. The CuiD shows 81% homology to YacK, a putative multicopper oxidases which extrudes copper in Escherichia coli. This ORF contains four conserved regions that contain 12 copper ligands (types 1, 2, and 3) present in various copper homeostasis responsible proteins. The H2O2 sensitive phenotype of the cuiD mutant indicates that cuiD may be involved in oxidative stress response.

The sctR of Salmonella enterica serova Typhimurium encoding a homologue of MerR protein is involved in the copper-responsive regulation of cuiD.

We have identified the cuiD gene in Salmonella enterica serova Typhimurium that codes for a putative multicopper oxidase. Expression of cuiD is induced by copper ion and its promoter/operator has sequence similarity to the promoters controlled by the transcriptional regulators of the MerR family. We also identified and isolated a gene from S. enterica serova Typhimurium that encodes a 138-amino acid residue protein, sctR, a new member of the MerR family of transcriptional regulators. Transposon-insertional disruption of sctR shows sensitivity to copper ion and no response of cuiD expression. Copper-responsive induction and copper tolerance were restored by providing sctR in trans, suggesting that SctR plays an important role in intracellular copper detoxification.