Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145-amino acid residues with extensive alpha-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.     

Induction of angiogenesis by expression of soluble type II transforming growth factor-beta receptor in mouse hepatoma.

The biological effect of transforming growth factor-beta (TGF-beta) is cell type-specific and complex. The precise role of TGF-beta is not clear in vivo. To elucidate the regulation mechanism of endogenous TGF-beta on hepatoma progression, we modified the MH129F mouse hepatoma cell with a retroviral vector encoding the extracellular region of type II TGF-beta receptor (TRII). Soluble TRII (TRIIs) blocked TGF-beta binding to TRII on the membrane of hepatoma cells. Growth of MH129F cells was inhibited by TGF-beta1 treatment; however, soluble TRII-overexpressing cells (MH129F/TRIIs) did not show any change in proliferation after TGF-beta1 treatment. MH129F/TRIIs cells also increased vascular endothelial growth factor (VEGF) expression, endothelial cell migration, and tube formation. Implantation of MH129F/TRIIs cells into C3H/He mice showed the significantly enhanced tumor formation. According to Western blot and protein kinase C assay, the expression of VEGF, KDR/flk-1 receptor, and endothelial nitric-oxide synthase was enhanced, and the phosphorylation activity of protein kinase C was increased up to 3.7-fold in MH129F/TRIIs tumors. Finally, a PECAM-1-stained intratumoral vessel was shown to be 4.2-fold higher in the MH129F/TRIIs tumor. These results indicate that VEGF expression is up-regulated by a blockade of endogenous TGF-beta signaling in TGF-beta-sensitive hepatoma cells and then stimulates angiogenesis and tumorigenicity. Therefore, we suggest that endogenous TGF-beta is a major regulator of the VEGF/flk-1-mediated angiogenesis pathway in hepatoma progression.     

Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay

Using 16S rRNA gene sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e., Campylobacter and Helicobacter) in red knot (Calidris canutus; n = 40), ruddy turnstone (Arenaria interpres; n = 35), and semipalmated sandpiper (Calidris pusilla; n = 22) fecal samples collected during a migratory stopover in Delaware Bay. Additionally, we studied the occurrence of Campylobacter spp., enterococci, and waterfowl fecal source markers using quantitative PCR (qPCR) assays. Of 3,889 16S rRNA clone sequences analyzed, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacteria (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). When epsilonproteobacterium-specific 23S rRNA gene clone libraries (i.e., 1,414 sequences) were analyzed, the sequences were identified as Campylobacter (82.3%) or Helicobacter (17.7%) spp. Specifically, 38.4%, 10.1%, and 26.0% of clone sequences were identified as C. lari (>99% sequence identity) in ruddy turnstone, red knot, and semipalmated sandpiper clone libraries, respectively. Other pathogenic species of Campylobacter, such as C. jejuni and C. coli, were not detected in excreta of any of the three bird species. Most Helicobacter-like sequences identified were closely related to H. pametensis (>99% sequence identity) and H. anseris (92% sequence identity). qPCR results showed that the occurrence and abundance of Campylobacter spp. was relatively high compared to those of fecal indicator bacteria, such as Enterococcus spp., E. faecalis, and Catellicoccus marimammalium. Overall, the results provide insights into the complexity of the shorebird gut microbial community and suggest that these migratory birds are important reservoirs of pathogenic Campylobacter species.     

Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes

A new medium (lecithin and levofloxacin [LL] medium) is described for the isolation of Listeria monocytogenes from food samples. LL medium includes lecithin from soybeans for the detection of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) produced by L. monocytogenes. Levofloxacin is incorporated to inhibit the growth of microorganisms other than L. monocytogenes, especially Bacillus cereus, shown to possess PI-PLC and PC-PLC activities. L. monocyogenes produced white colonies with a halo on LL medium, whereas Listeria innocua appeared as white colonies without a halo. Levofloxacin at 0.20 mg/liter completely inhibited the growth of B. cereus, while the growth of L. monocytogenes was unaffected. In the second phase of the study, the sensitivity and the specificity of LL medium were compared to those of modified Oxford agar (MOX) and two chromogenic media (Brilliance Listeria agar and CHROMagar Listeria), using a total of 250 food samples. From 200 unspiked food samples, the specificity of LL medium (96.0%) was superior to that of MOX (72.0%) and similar to the specificities of Brilliance Listeria agar (96.5%) and CHROMagar Listeria (94.5%). From 50 spiked food samples, LL medium and CHROMagar Listeria represented the highest sensitivities (96.0%), followed by Brilliance Listeria agar (92.0%) and MOX (54.0%). Also, LL medium showed the highest confirmation rate (98.8%), followed by Brilliance Listeria agar (98.7%), CHROMagar Listeria (98.3%), and MOX (52.0%). On the basis of its good specificity and cost effectiveness, LL medium is useful for the isolation of L. monocytogenes from food samples.     

Survival,prophage induction,and invasive properties of lysogenic Salmonella Typhimurium exposed to simulated gastrointestinal conditions

This study was designed to evaluate the viability, prophage induction, invasive ability, and relative gene expression in lysogenic Salmonella Typhimurium exposed to the simulated gastric juice (SGJ) at pH 2 (SGJ-2), 3 (SGJ-3), 4 (SGJ-4), and 5 (SGJ-5) for 30 min followed by 0.5 % bile salts for 2 h. The susceptibility of lysogenic S. Typhimurium increased with decreasing pH value and increasing bile salt concentration. The lysogenic S. Typhimurium cells were least susceptible to SGJ-4 and SGJ-5, showing <1 log reduction. The highest prophage induction was observed by 3.34 log PFU/ml in lysogenic S. Typhimurium at SGJ-3 in the presence of 0.5 % bile salts. The numbers of invading lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5 were 3.57, 3.73, and 4.15 log CFU/cm², respectively. Most genes (hilA, hilC, hilD, invA, invE, invF, and sirA) were down-regulated in lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5. This study provides useful information for understanding physiological changes of lysogenic S. Typhimurium in the simulated gastrointestinal conditions.     

Direct Measurements of Drag Forces in C. elegans Crawling Locomotion

With a simple and versatile microcantilever-based force measurement technique, we have probed the drag forces involved in Caenorhabditis elegans locomotion. As a worm crawls on an agar surface, we found that substrate viscoelasticity introduces nonlinearities in the force-velocity relationships, yielding nonconstant drag coefficients that are not captured by original resistive force theory. A major contributing factor to these nonlinearities is the formation of a shallow groove on the agar surface. We measured both the adhesion forces that cause the worm’s body to settle into the agar and the resulting dynamics of groove formation. Furthermore, we quantified the locomotive forces produced by C. elegans undulatory motions on a wet viscoelastic agar surface. We show that an extension of resistive force theory is able to use the dynamics of a nematode’s body shape along with the measured drag coefficients to predict the forces generated by a crawling nematode.     

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.     

Metabolomics investigation of flavonoid synthesis in soybean leaves depending on the growth stage

Soybean (Glycine max L.) leaves have unique nutraceutical and pharmacological benefits, and have been widely used as a source of healthy and functional food stuffs in Korea. In this study, we investigated the phytochemical metabolomic changes of soybean leaves depending on growth stages (maturation period) assessed based on UPLC–QTOF–MS analysis. Principal component analysis was carried out to trace the metabolite profiles of the phytochemicals from the vegetable stage (1D) through the seven reproductive stages (R1–R7). On the loading plot, significant changes in the contents of metabolites were found during the growth, and eight flavonoid kaempferol glycosides (2, 3, 6, 8, and 10), daidzein (14), genistein (17), and coumestrol (19) were evaluated as growth markers among the 19 isolated metabolites. The kaempferol glycosides were increasingly synthesized from the 1D to the R6 stage but decreased rapidly at stages R7–R8. The extensively synthesized daidzein and genistein were shown during seed growth in the pod (R5–R6), while coumestrol was increased significantly at stages R7–R8 (maturity period). The synthetic pathway of the flavonoids could be elucidated based on the concentration of the individual metabolites. These results demonstrate that the metabolite production changed depending on the growth stage; a possible pathway could be deduced using metabolomic analysis to provide information regarding physiological characterization and optimal harvesting time for crops.