Proteomic analysis of ganglioside‐associated membrane molecules: Substantial basis for molecular clustering

Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid‐enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X‐100 extracts and MS. We also examined GD3‐associated molecules using enzyme‐mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti‐GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3‐associating rafts exist. Representative examples of GD3‐associated molecules such as neogenin and MCAM were shown.     

Presence of two DNA polymerases in Tetrahymena pyriformis.

Two DNA polymerases were detected in Tetrahymena pyriformis, strain GL. One (enzyme I) was sensitive to N-ethylmaleimide, while the other (enzyme II) was insensitive. The molecular weight of the enzymes, as determined by glycerol gradient centrifugation analysis, were approximately 130,000 and 70,000, respectively. Optimal concentration of MgCl2 was 10mM for enzyme I and 18mM for enzyme II. KCl inhibited enzyme I but stimulated enzyme II. Poly (dA-dT) served effectively as a template for enzyme I, while poly(dA).(dT)12-18 was an effective template for enzyme II. Enzyme I activity increased with cell growth and sharply declined after the cells reached the stationary phase. On the other hand, enzyme II activity appeared only at the end of log phase. In cells synchronized by starvation-refeeding technique enzyme I was markedly stimulated in correspondence to the rate of DNA synthesis, whereas the level of enzyme II activity changed to lesser extent. By ethidium bromide treatment, only enzyme I activity was induced.     

Cell surface colony-stimulating factor 1 can be cleaved by TNF-alpha converting enzyme or endocytosed in a clathrin-dependent manner

CSF-1 is a hemopoietic growth factor, which plays an essential role in macrophage and osteoclast development. Alternative splice variants of CSF-1 are synthesized as soluble or membrane-anchored molecules, although membrane CSF-1 (mCSF-1) can be cleaved from the cell membrane to become soluble CSF-1. The activities involved in this proteolytic processing, also referred to as ectodomain shedding, remain poorly characterized. In the present study, we examined the properties of the mCSF-1 sheddase in cell-based assays. Shedding of mCSF-1 was up-regulated by phorbol ester treatment and was inhibited by the metalloprotease inhibitors GM6001 and tissue inhibitor of metalloproteases 3. Moreover, the stimulated shedding of mCSF-1 was abrogated in fibroblasts lacking the TNF-alpha converting enzyme (TACE, also known as a disintegrin and metalloprotease 17) and was rescued by expression of wild-type TACE in these cells, strongly suggesting that the stimulated shedding is TACE dependent. Additionally, we observed that mCSF-1 is predominantly localized to intracellular membrane compartments and is efficiently internalized in a clathrin-dependent manner. These results indicate that the local availability of mCSF-1 is actively regulated by ectodomain shedding and endocytosis. This mechanism may have important implications for the development and survival of monocyte lineage cells.     

Possible translocation of periodontal pathogens into the lymph nodes draining the oral cavity

Numerous publications have reported the presence of periodontopathogenic bacteria in peripheral and central vascular lesions. However, it is unclear how this bacterial translocation occurs. The objective of this study was to investigate whether periodontopathic bacteria are translocated to lymph nodes proximal to the oral cavity. Obtaining lymph node samples is not ethically feasible unless they are excised as part of the surgical management of patients with cancer. This study analyzed formalin-fixed and paraffin-embedded lymph nodes, histologically negative for cancer cell invasion, that were excised from 66 patients with histories of head and neck cancer. Real-time PCR was performed to amplify the 16S ribosomal DNA fragments from Porphyromonas gingivalis, Treponema denticola, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Prevotella intermedia. The relationship between bacterial detection and cancer severity, gender, and the use of anti-cancer therapy was examined by Fisher??s exact test. P. gingivalis, T. forsythia, and P. intermedia were present in 17%, 8%, and 8% of the samples of submandibular and submental lymph nodes, respectively. There were no significant relationships between bacterial detection and the cancer disease status, patient gender or use of anticancer therapy. According to these data, it appears that the translocation of periodontopathic bacteria may occur via lymphatic drainage, irrespective of the cancer disease status, gender or anticancer therapy.     

Transgenic expression of the EXT2 gene in developing chondrocytes enhances the synthesis of heparan sulfate and bone formation in mice

Hereditary multiple exostoses (HME), a dominantly inherited disorder characterized by multiple cartilaginous tumors, is caused by mutations in the gene for, EXT1 or EXT2. Recent studies have revealed that EXT1 and EXT2 are required for the biosynthesis of heparan sulfate and exert maximal transferase activity as a complex. The Drosophila homologue of EXT1 (tout-velu) regulates the movement and signaling of Hedgehog protein, which plays an important role in the regulation of chondrocyte differentiation and bone development. In this study, to investigate the biological role of EXT2 in bone development in vivo and the pathological role of HME mutations in the development of exostoses, we generated transgenic mice expressing EXT2 or mutant EXT2 in developing chondrocytes. Histological analyses and micro-CT scanning showed that the biosynthesis of heparan sulfate and the formation of trabeculae were upregulated in EXT2-transgenic mice, but not in mutant EXT2-transgenic mice. The expression of EXT1 is concomitantly upregulated in EXT2-transgenic and even mutant EXT2-transgenic mice, suggesting an interactive regulation of EXT1 and EXT2 expression. These findings support that the EXT2 gene encodes an essential component of the glycosyltransferase complex required for the biosynthesis of heparan sulfate, which may eventually modulate the signaling involved in bone formation.     

Role of the CM2 protein in the influenza C virus replication cycle

CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.     

Selenazoles (selenium compounds) facilitate survival of cultured rat pheochromocytoma PC12 cells after serum-deprivation and stimulate their neuronal differentiation via activation of Akt and mitogen-activated protein kinase, respectively

The activation of extracellular receptor kinase (ERK) is one of the checkpoints to assess the activation of the classical Ras/mitogen-activated protein kinase (MAPK) cascade. Therefore, we tested more than 100 selenium-containing compounds for their ability to activate the MAPK signal pathway. Among them, we found that three selenazoles, 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), 5-chloroacetyl-2-morpholino-1,3-selenazole (CS2), and 5-chloroacetyl-2-dimethylamino-1,3-selenazole (CS3), induced the phosphorylation of ERK. These compounds also enhanced the phosphorylation of Akt, a signal transducing protein kinase for cell survival; and this phosphorylation was followed by suppression of cell death, thus suggesting that they had anti-apoptotic effects. Moreover, CSs 1-3 induced neurite outgrowth and facilitated the expression of neurofilament-M of PC12 cells, demonstrating that they induced neuronal differentiation of these cells. On the other hand, the CS-induced phosphorylation of MAPK was enhanced by buthionine sulfoximine (BSO), an activator of protein tyrosine phosphatases (PTPs), but inhibited by N-acetyl-l-cysteine (NAC), an inhibitor of receptor tyrosine kinase. These results imply that activation of some receptor tyrosine kinase(s) is involved in the mechanism of action of CSs 1-3. The activation of MAPK by CSs 1-3 was suppressed by U0126, a MEK inhibitor, but not by K252a, an inhibitor of TrkA; AG1478, an antagonist of epidermal growth factor receptor (EGFR); or by pertussis toxin. These results demonstrate that the CS-induced phosphorylation of Akt and MAP kinase (receptor tyrosine kinase(s)-MEK1/2-ERK1/2) cascades was responsible for suppression of apoptosis and facilitation of neuronal differentiation of PC12 cells, respectively. Our results suggest that CSs 1-3 are promising candidates as neuroprotective and/or neurotrophic agents for the treatment of various neurodegenerative neurological disorders.     

Quantitative analysis of total serum glycome in human and mouse

Model mice are frequently used in drug discovery research. Knowledge of similarities and differences between the mouse and human glycomes is critical when model mice are used for the discovery of glycan‐related biomarkers and targets for therapeutic intervention. Since few comparative glycomic studies between human and mouse have been conducted, we performed a comprehensive comparison of the major classes of glycans in human and mouse sera using mass spectrometric and liquid chromatographic analyses. Up to 131 serum glycans, including N‐glycans, free oligosaccharides (fOSs), glycosaminoglycans, O‐glycans, and glycosphingolipid (GSL)‐glycans, were quantified. In both serum samples, N‐glycans were the most abundant in the total serum glycome, while fOSs were the least abundant. As expected, the diversity of sialic acid (i.e. Neu5Ac vs. Neu5Gc) was the major species difference between human and mouse in terms of N‐ and O‐glycosylation, while GSL‐glycomic profiles were completely different, even when the sialic acid diversity was taken into consideration. Furthermore, total serum glycomics of STAM mouse were unveiled as an initial step to identify novel biomarkers of liver diseases, with which we could identify several glycans with expression significantly increased or decreased expression.