Purification and Characterization of a Protein Cryoprotective for Vibrio cholerae Extracted from the Prawn Shell Surface

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti-CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.     

The Effects of Caffeine and Caffeine-containing Beverages on the Disposition of Brain Serotonin in Rats

Caffeine and caffeine-containing beverages (instant coffee, black tea, green tea, or oolong tea) caused a significant decrease in serum tryptophan, and significant increases in brain tryptophan, serotonin, and 5-hydroxyindole acetic acid over those in rats fed a control diet. Adenosine supplementation partially counteracted the increase of brain serotonin caused by caffeine. These results are interpreted as indicating that caffeine-containing beverages may have some nutritional and behavioral effects.     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.     

Neonatal lethality in knockout mice expressing the kinase-dead form of the gefitinib target GAK is caused by pulmonary dysfunction

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd(+/+) mice but not in GAK-kd(-/-) pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.     

Production of soluble matriptase by human cancer cell lines and cell surface activation of its zymogen by trypsin

The membrane-bound serine proteinase matriptase, which is often released from the plasma membrane of epithelial and carcinoma cells, has been implicated to play important roles in both physiological and pathological conditions. However, the regulatory mechanism of its activity is poorly understood. In the present study, we examined expression and activation state of soluble matriptase in 24 human cancer cell lines. Soluble matriptase was detected in the conditioned media from all of 5 colon and 4 breast carcinoma cell lines and 8 of 10 stomach carcinoma cell lines tested. Only two of five lung cancer cell lines released the matriptase protein into the culture media. Out of the five matriptase-negative cell lines, two cell lines expressed the matriptase mRNA. Among 24 cancer cell lines tested, 13 cell lines secreted trypsin in an active or latent form and all of them released matriptase. Most of the 24 cell lines released a latent, single-chain matriptase of 75 kDa as a major form, as well as low levels of complex forms of an activated two-chain enzyme with its specific inhibitor HAI-1. Thus, these soluble matriptases appeared to have little proteolytic activity. Treatment of stomach and colon cancer cell lines with epidermal growth factor stimulated the release of matripatase/HAI-1 complexes. In cancer cell lines secreting active trypsin, however, matriptase was released mostly as an inhibitor-free, two-chain active form. Trypsin seemed to activate the membrane-bound, latent matriptase on the cell surface. These results suggest that matriptase and trypsin cooperatively function for extracellular proteolysis.     

Epithelial-Mesenchymal Transition Stimulates Human Cancer Cells to Extend Microtubule-based Invasive Protrusions and Suppresses Cell Growth in Collagen Gel

Epithelial-mesenchymal transition (EMT) is a crucial event in tumor invasion and metastasis. However, most of past EMT studies have been conducted in the conventional two-dimensional (2D) monolayer culture. Therefore, it remains unclear what invasive phenotypes are acquired by EMT-induced cancer cells. To address this point, we attempted to characterize EMT cells in more physiological, three-dimensional (3D) collagen gel culture. EMT was induced by treating three human carcinoma cell lines (A549, Panc-1 and MKN-1) with TGF-ß. The TGF-ß treatment stimulated these cells to overexpress the invasion markers laminin γ2 and MT1-MMP in 2D culture, in addition to the induction of well-known morphological change and EMT marker expression. EMT induction enhanced cell motility and adhesiveness to fibronectin and collagen in 2D culture. Although EMT cells showed comparable cell growth to control cells in 2D culture, their growth rates were extremely suppressed in soft agar and collagen gel cultures. Most characteristically, EMT-induced cancer cells commonly and markedly extended invasive protrusions in collagen gel. These protrusions were mainly supported by microtubules rather than actin cytoskeleton. Snail-introduced, stable EMT cells showed similar protrusions in 3D conditions without TGF-ß. Moreover, these protrusions were suppressed by colchicine or inhibitors of heat shock protein 90 (HSP-90) and protein phosphatase 2A. However, MMP inhibitors did not suppress the protrusion formation. These data suggest that EMT enhances tumor cell infiltration into interstitial stroma by extending microtubule-based protrusions and suppressing cell growth. The elevated cell adhesion to fibronectin and collagen and high cell motility also seem important for the tumor invasion.     

GlmS and NagB regulate amino sugar metabolism in opposing directions and affect Streptococcus mutans virulence

Streptococcus mutans is a cariogenic pathogen that produces an extracellular polysaccharide (glucan) from dietary sugars, which allows it to establish a reproductive niche and secrete acids that degrade tooth enamel. While two enzymes (GlmS and NagB) are known to be key factors affecting the entrance of amino sugars into glycolysis and cell wall synthesis in several other bacteria, their roles in S. mutans remain unclear. Therefore, we investigated the roles of GlmS and NagB in S. mutans sugar metabolism and determined whether they have an effect on virulence. NagB expression increased in the presence of GlcNAc while GlmS expression decreased, suggesting that the regulation of these enzymes, which functionally oppose one another, is dependent on the concentration of environmental GlcNAc. A glmS-inactivated mutant could not grow in the absence of GlcNAc, while nagB-inactivated mutant growth was decreased in the presence of GlcNAc. Also, nagB inactivation was found to decrease the expression of virulence factors, including cell-surface protein antigen and glucosyltransferase, and to decrease biofilm formation and saliva-induced S. mutans aggregation, while glmS inactivation had the opposite effects on virulence factor expression and bacterial aggregation. Our results suggest that GlmS and NagB function in sugar metabolism in opposing directions, increasing and decreasing S. mutans virulence, respectively.     

Antibacterial activity of disodium succinoyl glycyrrhetinate,a derivative of glycyrrhetinic acid against Streptococcus mutans

Streptococcus mutans is a cariogenic bacterium that localizes in the oral cavity. Glycyrrhetinic acid (GRA) is a major component of licorice extract. GRA and several derivatives, including disodium succinoyl glycyrrhetinate (GR‐SU), are known to have anti‐inflammatory effects in humans. In this study, the antimicrobial effect of GRA and its derivatives against the S. mutans UA159 strain were investigated. Minimum inhibitory concentrations (MICs) of GRA and GR‐SU showed antibacterial activity against the S. mutans strain, whereas other tested derivatives did not. Because GR‐SU is more soluble than GRA, GR‐SU was used for further experiments. The antibacterial activity of GR‐SU against 100 S. mutans strains was evaluated and it was found that all strains are susceptible to GR‐SU, with MIC values below 256 µg/mL. A cell viability assay showed that GR‐SU has a bacteriostatic effect on S. mutans cells. As to growth kinetics, sub‐MICs of GR‐SU inhibited growth. The effect of GR‐SU on S. mutans virulence was then investigated. GR‐SU at sub‐MICs suppresses biofilm formation. Additionally, GR‐SU greatly suppresses the pH drop caused by the addition of glucose and glucose‐induced expression of the genes responsible for acid production (ldh and pykF) and tolerance (aguD and atpD). Additionally, expression of enolase, which is responsible for the carbohydrate phosphotransferase system, was not increased in the presence of GR‐SU, indicating that GR‐SU suppresses incorporation of sugars into S. mutans. In conclusion, GR‐SU has antibacterial activity against S. mutans and also decreases S. mutans virulence.     

Low-serum culture system improves the adipogenic ability of visceral adipose tissue-derived stromal cells

In obese adipose tissue, infiltrating macrophages release proinflammatory cytokines that trigger insulin resistance. An adipocyte-based platform from visceral fat would be useful to elucidate the pathology of adipose inflammation and to develop therapeutic drugs for insulin resistance. ADSCs (adipose tissue-derived mesenchymal stromal cells) expanded from subcutaneous fat are intensively studied as sources for regenerative medicine. However, the adipocyte culture system from visceral fat tissue has not been utilized yet. We aimed to establish the bioactive adipocyte platform using ADSCs from visceral fat pad. Stromal vascular fractions were processed from epididymal fat pads of Sprague-Dawley rats and three human omental fat pads, and the ADSCs were expanded using a low-serum culture method. The responses of ADSCs and ADSC-adipocytes (their adipogenic lineages) to pioglitazone, a therapeutic drug for diabesity, were evaluated by gene expression and ELISA. ADSCs (1×108) were expanded from 10 g of rat epididymal fat pads or human omental fat pads over five passages. Cell surface marker expressions revealed that visceral ADSCs were equivalent to mesenchymal stem cells. ADSC-adipocytes expanded in the low-serum culture system significantly showed higher expression of adipogenic markers [PPAR (peroxisome proliferator-activated receptor) γ, LPL (lipoprotein lipase) and FABP4 (fatty acid-binding protein 4)] and adipocytokines [adiponectin, resistin, leptin, PAI-1 (plasminogen-activator inhibitor 1) and IL (interleukin)-10] than those expanded in a high-serum culture system. Pioglitazone accelerated the adipogenic induction and increased adiponectin expression in human ADSCs by 57.9±5.8-fold (mean±S.E.M.) relative to control cells (P<0.001). Both in rat and human ADSC-adipocytes, TNF-α significantly induced proinflammatory cytokines [MCP-1 (monocyte chemoattractant protein-1) and IL-6] and suppressed adiponectin expression, while pioglitazone antagonized these effects. The present findings suggest that visceral ADSC-adipocytes expanded in low-serum culture would be useful for adiposcience and pharmacological evaluations.