Streptococcus pyogenes c-di-AMP Phosphodiesterase,GdpP, Influences SpeB Processing and Virulence

Small cyclic nucleotide derivatives are employed as second messengers by both prokaryotes and eukaryotes to regulate diverse cellular processes responding to various signals. In bacteria, c-di-AMP has been discovered most recently, and some Gram-positive pathogens including S. pyogenes use this cyclic nucleotide derivative as a second messenger instead of c-di-GMP, a well-studied important bacterial second messenger. GdpP, c-di-AMP phosphodiesterase, is responsible for degrading c-di-AMP inside cells, and the cellular role of GdpP in S. pyogenes has not been examined yet. To test the cellular role of GdpP, we created a strain with a nonpolar inframe deletion of the gdpP gene, and examined the properties of the strain including virulence. From this study, we demonstrated that GdpP influences the biogenesis of SpeB, the major secreted cysteine protease, at a post-translational level, susceptibility to the beta lactam antibiotic ampicillin, and is necessary for full virulence in a murine subcutaneous infection model.     

Perforator-based flap in rats: a new experimental model.

A new type of flap, the perforator-based flap, has been described in the last decade. It has been used successfully as a pedicle or free flap by many plastic surgeons. There is no animal model for research, although these flaps have gained popularity in clinical use. We created a perforator-based flap model in the rat (a perforator-based flap group and two control groups; 10 rats in each group) and evaluated the survival characteristics of the new flap. The abdominal skin flap was elevated based on the second perforator of the right superior deep epigastric artery and then sutured to its original bed. In the first control group, the same flap was elevated with a subcutaneous pedicle without any perforator; in the second control group, a right-sided, random-pattern pedicle abdominal skin flap with the same dimensions and location was elevated and sutured to its original bed. Flap survival was studied, and microangiography and histologic studies were performed. The amount of viable skin in the three groups was compared 1 week later. The area of surviving skin paddles in the experimental group ranged from 74 to 83 percent; in the first control group, it was 0 percent; and in the second control group, it ranged from 29 to 44 percent (p < 0.001 and p < 0.001, respectively). There was a predictable and constant area of necrosis in the model.The results of this study demonstrate that most of the abdominal skin of the rat can survive on the basis of a single musculocutaneous perforator vessel. This flap can be easily elevated, and it can be used as a reliable model for flap research.     

Design,Synthesis, and Anti-Tumor Activity of 4′-Thionucleosides as Potent and Selective Agonists at the Human A3 Adenosine Receptor

On the basis of potent and selective binding affinity of Cl-IB-MECA to the human A₃ adenosine receptor, its 4′-thioadenosine derivatives were efficiently synthesized starting from D-gulonic γ -lactone. Among compounds tested, 2-chloro-N ⁶-(3-iodobenzyl)- and 2-chloro-N ⁶-methyl-4′ -thioadenosine-5′ -methyluronamides (7a and 7b) exhibited nanomolar range of binding affinity (K _i = 0.38 nM and 0.28 nM, respectively) at the human A₃AR. These compounds showed anti-growth effects on HL-60 leukemia cell, which resulted from the inhibition of Wnt signaling pathway.     

Capsaicin induces apoptosis by generating reactive oxygen species and disrupting mitochondrial transmembrane potential in human colon cancer cell lines

Although genetic factors are a well-known cause of colorectal cancer, environmental factors contribute more to its development. Despite advances in the fields of surgery, radiotherapy and chemotherapy, the cure rates for colon cancer have not substantially improved over the past few decades. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the principal pungent ingredient of hot chili pepper, has exhibited an anti-tumor effect in many cell types. However, the mechanisms responsible for the anti-tumor effect of capsaicin are not yet completely understood. In this study, we investigated whether capsaicin induces apoptosis in colon cancer cell lines. Capsaicin decreased cell viability in a dose-dependent manner in Colo320DM and LoVo cells. In addition, capsaicin produced cell morphology changes and DNA fragmentation, decreased the DNA contents, and induced phosphatidylserine translocation, which is a hallmark of apoptotic cell death. We showed that capsaicin-induced apoptosis is associated with an increase in ROS generation and a disruption of the mitochondrial transmenbrane potential. A possible mechanism of capsaicin-induced apoptosis is the activation of caspase 3, a major apoptosis-executing enzyme. Treatment with capsaicin induced a dramatic increase in caspase 3 activity, as assessed by the cleavage of Ac-DEVD-AMC, a fluorogenic substrate. In conclusion, our results clearly showed that capsaicin induced apoptosis in colon cancer cells. Although the actual mechanisms of capsaicin-induced apoptosis remain uncertain, it may be a beneficial agent for colon cancer treatment and chemoprevention.     

Identification of Differentially Expressed Genes in Flower Buds of <Emphasis Type="Italic">Calanthe discolor</Emphasis> and <Emphasis Type="Italic">C. sieboldii</Emphasis>

The genus Calanthe includes species of terrestrial orchids that produce attractive flowers with diverse floral traits. Breeding programs have been established to improve the horticultural value of various Calanthe species, but studies to identify the genetic components contributing to the key phenotypic characteristics have not been undertaken. To understand the molecular mechanisms underlying floral development associated with floral morphology, color, and fragrance production, the flower buds of two typical Korean Calanthe species, C. discolor and C. sieboldii, were subjected to gene expression analysis by differential display RT-PCR (DDRT-PCR). A total of 66 non-redundant differentially expressed genes (DEGs) were isolated and sequenced. Of these, 26 and 40 DEGs were found to be highly expressed in C. discolor and C. sieboldii, respectively. Moreover, the expression patterns of a subset of genes presumably implicated in signal transduction, metabolic pathways, and hormonal signaling differed between the two species. The data presented here may improve our understanding of the mechanisms underlying floral development and contribute to advances in orchid biotechnology.     

ISG15 modification of filamin B negatively regulates the type I interferon‐induced JNK signalling pathway

Interferon (IFN)‐induced signalling pathways have essential functions in innate immune responses. In response to type I IFNs, filamin B tethers RAC1 and a Jun N‐terminal kinase (JNK)‐specific mitogen‐activated protein kinase (MAPK) module—MEKK1, MKK4 and JNK—and thereby promotes the activation of JNK and JNK‐mediated apoptosis. Here, we show that type I IFNs induce the conjugation of filamin B by interferon‐stimulated gene 15 (ISG15). ISGylation of filamin B led to the release of RAC1, MEKK1 and MKK4 from the scaffold protein and thus to the prevention of sequential activation of the JNK cascade. By contrast, blockade of filamin B ISGylation by substitution of Lys 2467 with arginine or by knockdown of ubiquitin‐activating enzyme E1‐like (UBEL1) prevented the release of the signalling molecules from filamin B, resulting in persistent promotion of JNK activation and JNK‐mediated apoptosis. These results indicate that filamin B ISGylation acts as a negative feedback regulatory gate for the desensitization of type I IFN‐induced JNK signalling.     

Gram‐positive bacteria produce membrane vesicles: Proteomics‐based characterization of Staphylococcus aureus‐derived membrane vesicles

Although archaea, Gram‐negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell‐free intercellular communication, this cellular process has been overlooked in Gram‐positive bacteria. Here, we found for the first time that Gram‐positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus‐derived MVs are both similar to those of Gram‐negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus‐derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram‐positive bacteria.