Increased cyclin B1/CDC 2 kinase activity and phosphorylation of Bcl-2 associated with paclitaxel-induced apoptosis in human nasopharyngeal carcinoma cells

Paclitaxel is a potential cancer chemotherapeutic agent for ovary, breast, and head and neck cancers; its effects on nasopharyngeal carcinoma (NPC) have not been reported previously. This study investigated the cytotoxic mechanism of paclitaxel in two NPC cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with pacli-taxel showed convoluted nuclei, condensed chromatin and decreased cellular and nuclear volume, and also exhibited genomic DNA degradation into multiple oligonucleosomal fragments, suggesting that pacli-taxel induced apoptosis in these cells. The effects of paclitaxel on apoptosis-related proteins including Bcl-2, Bax and CDC 2 were also detected. Although the levels of Bcl-2 and Bax were not changed in NPC cells following treatment with 5 nM-1 μM of paclitaxel, phosphorylation of Bcl-2 was significantly observed in the cells treated with 1 μM of paclitaxel for 12 hours. In addition, cyclin B1-associated CDC 2 kinase was highly activated in the NPC cells exposed to paclitaxel even at low (5 nM) concentration, and this result is associated with the finding that low concentration of paclitaxel is able to induce apoptosis in NPC cells.     

C-terminus of Hsc70-interacting protein (CHIP) enhances stemness properties of human Wharton’s jelly mesenchymal stem cell

Mesenchymal stem cells are an attractive source of multipotent cells in part because they are easy to obtain. Several E3 ligases regulate the stability and functions of various factors in different adult stem cells through the ubiquitylation pathway. We investigated the C-terminus of Hsc70-interacting protein (CHIP) E3 ligase that regulates pluripotency of human Wharton’s jelly mesenchymal stem cells (hWJMSC). We found that CHIP increases protein kinase B (Akt) phosphorylation by decreased expression of phosphatase and tensin homolog (PTEN), which suggests improvement of the survival pathway by CHIP over-expression. We also found that increased CHIP expression induced Sox2 and NANOG, which can promote stem cell self-renewal and prevent oxidative stress-induced senescence of hWJMSC by decreased p21. We found that CHIP could be used to enhance the multiple functions of hWJMSC.     

Mouse inbred strain differences in ethanol drinking to intoxication

Recently, we described a simple procedure, Drinking in the Dark (DID), in which C57BL/6J mice self-administer ethanol to a blood ethanol concentration (BEC) above 1 mg/ml. The test consists of replacing the water with 20% ethanol in the home cage for 4 h early during the dark phase of the light/dark cycle. Three experiments were conducted to explore this high ethanol drinking model further. In experiment 1, a microanalysis of C57BL/6J behavior showed that the pattern of ethanol drinking was different from routine water intake. In experiment 2, drinking impaired performance of C57BL/6J on the accelerating rotarod and balance beam. In experiment 3, 12 inbred strains were screened to estimate genetic influences on DID and correlations with other traits. Large, reliable differences in intake and BEC were detected among the strains, with C57BL/6J showing the highest values. Strain means were positively correlated with intake and BEC in the standard (24 h) and a limited (4 h) two-bottle ethanol vs. water test, but BECs reached higher levels for DID. Strain mean correlations with other traits in the Mouse Phenome Project database supported previously reported genetic relationships of high ethanol drinking with low chronic ethanol withdrawal severity and low ethanol-conditioned taste aversion. We extend these findings by showing that the correlation estimates remain relatively unchanged even after correcting for phylogenetic relatedness among the strains, thus relaxing the assumption that the strain means are statistically independent. We discuss applications of the model for finding genes that predispose pharmacologically significant drinking in mice.     

Computational aspects of protein functionality

The purpose of this short article is to examine certain aspects of protein functionality with relation to some key organizing ideas. This is important from a computational viewpoint in order to take account of modelling both biological systems and knowledge of these systems. We look at some of the lexical dimensions of the function and how certain constructs can be related to underlying ideas. The pervasive computational metaphor is then discussed in relation to protein multifunctionality, and the specific case of von Willebrand factor as a 'smart' multifunctional protein is briefly considered. Some diagrammatic techniques are then introduced to better articulate protein function.     

Glucooligosaccharides from <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> B-742 (ATCC 13146): A potential prebiotic

There is an emerging market for functional oligosaccharides for use in foods. Currently, technology for the production of oligosaccharides is limited to extraction from plant sources, acid or enzymatic hydrolysis of polysaccharides or synthesis by transglycosylation reactions. Oligosaccharides can also be produced using a Leuconostoc fermentation and restricting the polymer size by addition of maltose. Maltose limits the dextransucrase reaction, yielding high concentrations of α-glucooligosaccharides. Branched oligomers produced by this process were readily catabolized by bifidobacteria and lactobacilli but were not readily utilized by either Salmonella sp. or Escherichia coli, pointing toward their use in intestinal microflora modification. Journal of Industrial Microbiology & Biotechnology (2002) 29, 196–199 doi:10.1038/sj.jim.7000269 Received 13 February 2002/ Accepted in revised form 29 April 2002     

Structural and expression analyses of two vitellogenin genes in the carp, Cyprinus carpio

We cloned and sequenced two vitellogenin (vg) cDNAs of the carp, Cyprinus carpio, using a cDNA library constructed from estradiol-17β (E₂)-treated livers. One was a novel, longer 5000 bp-long cDNA termed vg-B2 encoding 1624 amino acids in a single open reading frame. The other was a shorter cDNA (vg-B1), identical to that registered previously as carp vg cDNA in the international nucleotide sequence database. The deduced amino acid sequences of these two molecules were well-aligned with known vertebrate Vgs sharing common characteristics such as N-terminal lipovitellin I (LVI), phosvitin (PV) and C-terminal lipovitellin II (LVII). The novel Vg-B2 bore a highly conserved GL/ICG motif within the LVII region, in contrast to the shorter Vg-B1 that has a truncated C-terminal and lacks the β-component within the LVII region including the GL/ICG motif. Both vg-B2 and vg-B1 genes were expressed in the livers of females and E₂-injected males. Western blot analysis using anti-Vg and anti-vitellin (Vn) antisera demonstrated that both Vg-B2 and Vg-B1 were detected as polypeptides with an estimated molecular mass of 180 kDa and 160 kDa, respectively, in the blood of females and E₂-injected males. The results suggest the potential utilization of these genes as sensitive xenoestrogenic markers.     

LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14⁺TLR4⁻, whereas cancerous tissues were CD14⁺TLR4⁺, by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14⁺TLR4⁺ colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14⁺TLR4⁻) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.Colorectal tumorigenesis proceeds via the accumulation of genetic and epigenetic alterations that promote unlimited cell proliferation, self-sufficient growth signaling, neovascularization, tissue invasion, and resistance to cell death.¹ The transformation of normal epithelium into colorectal carcinomas (CRC) is associated with the progressive inhibition of apoptosis; this confers a selective advantage for tumor cell survival and chemoresistance.^{2, 3} It is generally believed that sufficient epithelial apoptosis may hamper colon cancer formation in terms of incidence and growth rate.^{4, 5, 6} Direct evidence for this was recently reported in mice deficient in pro-apoptotic molecules.^{7, 8} To date, the regulatory mechanisms of physiological apoptosis to eliminate premalignant cells in the gut remain incompletely understood.Intestinal homeostasis is maintained by the dynamic, yet strictly regulated, turnover of epithelial cells. An imbalance in epithelial death versus survival/proliferative responses may lead to barrier dysfunction, chronic inflammation, and tumorigenesis.^{9, 10} Accumulating evidence indicates that gut microbiota and bacterial lipopolysaccharide (LPS) have critical roles in epithelial cell renewal under baseline conditions and on injury,^{11, 12} and are involved in the pathogenesis of colitis-associated CRC as well.^{13, 14, 15} Given the juxtaposition of commensal bacteria and the gut mucosa, it has been assumed that normal epithelial cells are not equipped with LPS receptor complexes (CD14/TLR4/MD2) or express altered forms of receptors and signaling molecules to achieve immunotolerance.¹⁵ Constitutive expression of CD14 was reported in the presence of negligible-to-low levels of Toll-like receptor 4 (TLR4) in normal human colonocytes,^{16, 17, 18} whereas strong TLR4 immunoreactivity was detected in CRC.^{18, 19} Nevertheless, divergent cellular responses to LPS (death versus survival) have been reported among human CRC cell lines. Several laboratories, using Caco-2 cells, have described increases in apoptotic cell death following apical LPS challenge,^{20, 21} whereas others have documented enhanced survival and proliferative responses of HT29 and SW480 cells to LPS.^{22, 23} Here we hypothesize that differing expression patterns of LPS receptor subunits on epithelial surfaces may have a determining role in cell death versus survival.CD14, as the membrane-bound subunit of LPS receptor complex and lacking a cytoplasmic tail, has traditionally been regarded as merely a binding component for transferring LPS to TLR4. TLR4 subsequently activates downstream adaptors and signaling pathways, such as myeloid differentiation factor (MyD88), mitogen-activated protein kinases (MAPKs), inhibitor of κB (IκB)/nuclear factor-κB (NFκB), and interferon regulatory factor 3 (IRF3).^{24, 25} Recent findings in monocytes have indicated that LPS/CD14 binding triggers a cascade of lipid messenger signals before TLR4 trafficking to lipid rafts for complex formation. CD14-dependent lipid signaling includes the conversion of membranous phosphatidylcholine (PC) to diacylglcerol by PC-specific phospholipase C (PC-PLC) and the activation of sphingomyelinase (SMase) for sphingolipid metabolism and ceramide production. This process leads to the phosphorylation of protein kinase C (PKC) ζ, which recruits TLR4 to interact with CD14 (Cuschieri et al.²⁶ and Triantafilou et al.²⁷). Lipid messengers, such as sphingolipids and ceramides, and their downstream PKCζ signals have been implicated in pro-apoptotic pathways and are considered tumor suppressors.^{28, 29, 30} Decreased SMase activity and PKCζ levels have been observed in human colorectal tumors, correlated with poor prognosis.^{31, 32} In contrast, the TLR4/MyD88 and IκB/NFκB pathways are associated with anti-apoptotic and hyperproliferative responses.^{5, 33, 34, 35} Reduced colorectal tumor formation has been documented in TLR4(−/−), MyD88(−/−), and epithelial-specific IκB kinase β-deficient mice as compared with wild-type (WT) mice.^{5, 19, 36} These findings led us to speculate that the expression of CD14 and TLR4 on epithelial cell surfaces may provide antagonistic signals to counteract apoptotic responses to LPS and to influence tumor progression.The aims of this study were to (1) investigate the expression patterns of LPS receptor subunits in normal and cancerous colonic epithelia in human and murine tissues; (2) examine the individual roles of CD14 and TLR4 in epithelial apoptosis and tumor formation using a mouse model of colitis-associated CRC; (3) assess the involvement of CD14-mediated lipid messengers and/or TLR4-dependent signaling in the mechanism of LPS-induced apoptosis using human carcinoma cell lines; and (4) evaluate whether TLR4 has an opposing role against CD14-mediated apoptosis to promote tumor cell survival.