Proteomic analysis of outer membrane vesicles derived from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, constitutively secretes outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress has revealed that OMVs are essential for pathogenesis of P. aeruginosa, their proteins have not been comprehensively analyzed so far. In this study, we identified 338 vesicular proteins with high confidence by five separate LC-MS/MS analyses. This global proteome profile provides a basis for future studies to elucidate the pathological functions of OMVs from P. aeruginosa.     

The protein interaction network of extracellular vesicles derived from human colorectal cancer cells

Various mammalian cells including tumor cells secrete extracellular vesicles (EVs), otherwise known as exosomes and microvesicles. EVs are nanosized bilayered proteolipids and play multiple roles in intercellular communication. Although many vesicular proteins have been identified, their functional interrelationships and the mechanisms of EV biogenesis remain unknown. By interrogating proteomic data using systems approaches, we have created a protein interaction network of human colorectal cancer cell-derived EVs which comprises 1491 interactions between 957 vesicular proteins. We discovered that EVs have well-connected clusters with several hub proteins similar to other subcellular networks. We also experimentally validated that direct protein interactions between cellular proteins may be involved in protein sorting during EV formation. Moreover, physically and functionally interconnected protein complexes form functional modules involved in EV biogenesis and functions. Specifically, we discovered that SRC signaling plays a major role in EV biogenesis, and confirmed that inhibition of SRC kinase decreased the intracellular biogenesis and cell surface release of EVs. Our study provides global insights into the cargo-sorting, biogenesis, and pathophysiological roles of these complex extracellular organelles.     

Specific interaction of VEGF165 with beta-amyloid, and its protective effect on beta-amyloid-induced neurotoxicity

beta-amyloid (Abeta) is a major component of senile plaques that is commonly found in the brain of Alzheimer's disease (AD) patient. In the previous report, we showed that an important angiogenic factor, vascular endothelial growth factor (VEGF) interacts with Abeta and is accumulated in the senile plaques of AD patients' brains. Here we show that Abeta interacts with VEGF(165) isoform, but not with VEGF(121). Abeta binds to the heparin-binding domain (HBD) of VEGF(165) with similar affinity as that of intact VEGF(165). Abeta binds mostly to the C-terminal subdomain of HBD, but with greatly reduced affinity than HBD. Therefore, the full length of HBD appears to be required for maximal binding of Abeta. Although Abeta binds to heparin-binding sequence of VEGF, it does not bind to other heparin-binding growth factors except midkine. Thus it seems that Abeta recognizes unique structural features of VEGF HBD. VEGF(165) prevents aggregation of Abeta through its HBD. We localized the core VEGF binding site of Abeta at around 26-35 region of the peptide. VEGF(165) and HBD protect PC12 cells from the Abeta-induced cytotoxicity. The mechanism of protection appears to be inhibition of both Abeta-induced formation of reactive oxygen species and Abeta aggregation.     

Human CC chemokine CCL23, a ligand for CCR1, induces endothelial cell migration and promotes angiogenesis

A number of chemokines induce angiogenesis and endothelial cells express several chemokine receptors. To date, only a limited number of CC chemokines for CCR1 have been reported to induce angiogenic responses. We investigated the ability of CCL23 (also known as MPIF-1, MIP-3, or CKbeta8) to promote angiogenesis, which induces chemotaxis of immune cells through CCR1. CCL23 promoted the chemotactic migration and differentiation of endothelial cells, and neovascularization in the chick chorioallantoic membrane. An N-terminal truncated form of CCL23 was at least 100-fold more potent than its intact form and was comparable to that of FGF in the angiogenic activities. Treatment with either pertussis toxin or anti-CCR1 antibody completely inhibited the CCL23-induced endothelial cell migration, indicating that endothelial cell migration was mediated through CCR1. CCL23 didn't promote the migration of HT1080 human fibrosarcoma cells that did not express CCR1. Our results suggest a role of CCL23 in angiogenesis in vitro as well as in vivo.     

Deletions fusing the i and lac regions of the chromosome in E. coli: isolation and mapping

Summary Three hundred and fifty deletions ending within the i gene (which codes for the lac repressor) at one terminus and within the z gene at the other terminus have been isolated. Twenty-nine of these have been mapped in detail and have endpoints which are distributed throughout the i gene. Two deletions which remove only the very end of the i gene still permit the synthesis of altered repressor molecules retaining some properties of the wildtype lac repressor. In strains carrying these deletions, the synthesis of lac permease is under the control of the i promoter. An additional 40 deletions have been found which apparently fuse the lac permease to an untranslated portion of the terminus of the i-message.     

Luteinizing hormone releasing hormone-RNase A conjugates specifically inhibit the proliferation of LHRH-receptor-positive human prostate and breast tumor cells

Human prostate and breast tumor cells produce luteinizing hormone-releasing hormone (LHRH) receptors on their cell surface even when they have lost dependency on sex steroid hormones for growth. To investigate whether LHRH can be used as a cell-binding moiety to deliver toxin molecules into prostate and breast tumor cells, LHRH-bovine RNase A conjugates were constructed using the chemical cross-linking method. The treatment of the LHRH receptor-positive cells such as prostate LNCapFGC and breast MCF7 tumor cells with LHRH-RNase A conjugates resulted in a dose-dependent inhibition of growth. The cytotoxic activities of these conjugates were effectively reduced by the presence of exogenous LHRH. Either free RNase A or LHRH alone did not affect the proliferation of these cells. The LHRH-RNase A conjugates did not show cytotoxicity against FRTL5 and TM4 cells which do not express the LHRH receptors. These results suggest that LHRH can be used as a cell-binding molecule for the specific delivery of toxin molecules into the cells which express LHRH receptors on their surface. Thus, a new class of biomedicines that act as fusion proteins between LHRH and toxins will give us a new avenue for the treatment of human prostate and breast cancers, regardless of their steroid hormone dependency.     

Arginine-rich anti-vascular endothelial growth factor peptides inhibit tumor growth and metastasis by blocking angiogenesis

Tumor angiogenesis is a critical step for the growth and metastasis of solid tumors. Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and contributes to the development of solid tumors by promoting tumor angiogenesis. Therefore, it is a prime therapeutic target for the development of antagonists for treatment of cancer. We identified from peptide libraries arginine-rich hexapeptides that inhibit the interaction of VEGF(165) with VEGF receptor (IC(50) = 2-4 micrometer). They have no effect on binding of basic fibroblast growth factor to cellular receptor. The hexapeptides inhibit the proliferation of human umbilical vein endothelial cells induced by VEGF(165) without toxicity. The peptides bind to VEGF and inhibit binding of both VEGF(165) and VEGF(121), suggesting that the peptides interact with the main body of VEGF but not the heparin-binding domain that is absent in VEGF(121). The identified peptides block the angiogenesis induced by VEGF(165) in vivo in the chick chorioallantoic membrane and the rabbit cornea. Furthermore, one of the hexapeptides, RRKRRR, blocks the growth and metastasis of VEGF-secreting HM7 human colon carcinoma cells in nude mice. Based on our results, the arginine-rich hexapeptides may be effective for the treatment of various human tumors and other angiogenesis-dependent diseases that are related to the action of VEGF and could also serve as leads for development of more effective drugs.     

A quantitative assessment of the dependency of the visual temporal frame upon the cortical rhythm

This paper reports an attempt to measure the dependency of the visual temporal frame on cortical rhythms. As a probe, we used two short flashes of a point light source separated in time by a variable interstimulus interval (ISI). Subjects were asked to judge whether the two short flashes were perceived as simultaneous or as sequential. The onset of the light flashes was triggered at four chosen phases of the occipital alpha rhythm of the viewing subject, and the probabilities of perceived simultaneity at various ISI's were obtained. Data from three experimental subjects show that the triggering phase produced a significant shift along the ISI-axis, thus changing the probability of perceived simultaneity. The magnitude of this shift varied substantially across subjects. These results show that, although cortical rhythms correlate with perceptual framing as previously reported (Varela et al., 1981), the nature of this dependency is different than previously suggested: a visual temporal frame seems to be a local event at the cortex, and not a global coordination uniformly reflected in the alpha rhythm.     

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.