Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.     

A highly sensitive high-performance liquid chromatography-- fluorometric method for the assay of peptidylarginine deiminase activity

The activity of peptidylarginine deiminase (PAD) has generally been assayed by a colorimetric method using N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine (Bz-L-Arg) as the substrates. The widespread occurrence of citrulline and urea in tissues makes use of this method difficult, especially for small samples. We developed a highly sensitive high-performance liquid chromatography method with N-dansyl-glycyl-L-arginine as the substrate. This method was sensitive enough to determine previously undetectable activity of PAD in HL-60 cells. Two types of PAD (HL-60 cell and brain PAD) could be distinguished by differential competition, using either BAEE or Bz-L-Arg as a preferential substrate in the assay. These data indicate that the present method is applicable to many tissues.     

Four alkaloids, lucidine B, oxolucidine A, lucidine A, and lucidulinone from Lycopodium lucidulum

The structures of four alkaloids extracted from Lycopodium lucidulum (Lycopodiaceae) were established by X-ray and 2D NMR spectroscopic analyses. The dihydro-derivative of oxolucidine A, which was obtained by NaBH4 reduction of oxolucidine A, was treated with p-bromobenzoyl chloride to afford crystals, whose X-ray crystallographic analysis established the stereostructure, including the absolute configuration. The 2D NMR spectra of tetrahydrodeoxylucidine B were fully analyzed to establish the full structure of lucidine B, and the hitherto unknown stereochemistry at the C-14 position was established as beta-H. The structure of a new alkaloid, lucidulinone, was determined by spectroscopic analysis to be luciduline lactam.     

Autolysis during in vitro tracheary element differentiation: formation and location of the perforation

Tracheary elements differentiated from isolated Zinnia: mesophyll cells were observed at various times of culture under a scanning electron microscope. Perforation occurred on the primary wall at one of the longitudinal ends in single tracheary elements. In double tracheary elements, which both of two cells derived from a single cell differentiated into, the pore opened on the primary walls both at the junction of the two tracheary elements and at a longitudinal end of one of the two tracheary elements. These results suggest not only that a single tracheary element has its own program to form a perforation at one end without being affected by neighboring cells, but also that isolated cells indeed hold some traces of polarity and cell-cell communication.     

MAPKKK-Related protein kinase NPK1: Regulation of the M phase of plant cell cycle

The tobaccoNPK1 gene encodes a homolog of mitogenactivated protein kinase kinase kinases. We have recently identified tobacco kinesin-like proteins (NACK1/2) as activators for NPK1. Immunochemical analyses of NPK1 and NACK1 proteins suggest that NPK1 is involved in the regulation of some process in the M phase of the plant cell cycle. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Predicting the response of the deep-ocean microbiome to geochemical perturbations by hydrothermal vents

Submarine hydrothermal vents perturb the deep-ocean microbiome by injecting reduced chemical species into the water column that act as an energy source for chemosynthetic organisms. These systems thus provide excellent natural laboratories for studying the response of microbial communities to shifts in marine geochemistry. The present study explores the processes that regulate coupled microbial-geochemical dynamics in hydrothermal plumes by means of a novel mathematical model, which combines thermodynamics, growth and reaction kinetics, and transport processes derived from a fluid dynamics model. Simulations of a plume located in the ABE vent field of the Lau basin were able to reproduce metagenomic observations well and demonstrated that the magnitude of primary production and rate of autotrophic growth are largely regulated by the energetics of metabolisms and the availability of electron donors, as opposed to kinetic parameters. Ambient seawater was the dominant source of microbes to the plume and sulphur oxidisers constituted almost 90% of the modelled community in the neutrally-buoyant plume. Data from drifters deployed in the region allowed the different time scales of metabolisms to be cast in a spatial context, which demonstrated spatial succession in the microbial community. While growth was shown to occur over distances of tens of kilometers, microbes persisted over hundreds of kilometers. Given that high-temperature hydrothermal systems are found less than 100 km apart on average, plumes may act as important vectors between different vent fields and other environments that are hospitable to similar organisms, such as oil spills and oxygen minimum zones.     

A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.     

Des-γ-Carboxyprothrombin (DCP) and NX-DCP Expressions and Their Relationship with Clinicopathological Features in Hepatocellular Carcinoma

Aim

Des-γ-carboxyprothrombin (DCP) has been used as a tumor marker for hepatocellular carcinoma (HCC). Recently the DCP/NX-DCP ratio, calculated by dividing DCP by NX-DCP, has been reported useful in detecting HCC. The purpose of this study is to clarify the significance of DCP and NX-DCP expression in HCC tissues.

Methods

HCC and non-HCC tissue samples were obtained from 157 patients and were immunohistochemically examined for DCP and NX-DCP expression using anti-DCP antibody and anti-NX-DCP antibody. DCP and NX-DCP expression scores were calculated by multiplying staining intensity grade by percentage of stained area. Serum DCP and NX-DCP levels were determined in 89 patients. We evaluated the relationship between tumor expression, serum level, and pathomorphological findings.

Results

Intrahepatic metastasis (im) was significantly more frequent in cases with high DCP expression than in cases with low DCP expression. High NX-DCP expression was associated with significantly lower histological grade, and less frequent im or portal vein invasion (vp) than low NX-DCP expression. Serum DCP was correlated with DCP expression, but serum NX-DCP was not correlated with NX-DCP expression. DCP-positive (≥40 mAU/L), NX-DCP-positive (≥90 mAU/L), and DCP/NX-DCP ratio-positive (≥1.5) cases were associated with significantly larger tumor size and more frequent vp than negative cases. DCP was rarely expressed, but NX-DCP was frequently expressed in non-cancerous liver tissues. Patients with NX-DCP expression-negative tumors showed a lower survival rate than those with NX-DCP expression-positive tumors (p = 0.04), whereas the survival in serum NX-DCP-positive cases was lower than that of serum negative cases (p = 0.02).

Conclusions

DCP and NX-DCP were produced in HCC tissues, but differed in expression level and biological properties. DCP expression, serum DCP or NX-DCP level, and DCP/NX-DCP ratio were closely related to malignant properties of HCC.     

Induction of CD44 Variant 9-Expressing Cancer Stem Cells Might Attenuate the Efficacy of Chemoradioselection and Worsens the Prognosis of Patients with Advanced Head and Neck Cancer

Background

At our institute, a chemoradioselection strategy has been used to select patients for organ preservation on the basis of response to an initial 30–40 Gy concurrent chemoradiotherapy (CCRT). Patients with a favorable response (i.e., chemoradioselected; CRS) have demonstrated better outcomes than those with an unfavorable response (i.e., nonchemoradioselected; N-CRS). Successful targeting of molecules that attenuate the efficacy of chmoradioselection may improve results. Thus, the aim of this study was to evaluate the association of a novel cancer stem cell (CSC) marker, CD44 variant 9 (CD44v9), with cellular refractoriness to chemoradioselection in advanced head and neck squamous cell carcinoma (HNSCC).

Materials and Methods

Through a medical chart search, 102 patients with advanced HNSCC treated with chemoradioselection from 1997 to 2008 were enrolled. According to our algorithm, 30 patients were CRC following induction CCRT and 72 patients were N-CRS. Using the conventional immunohistochemical technique, biopsy specimens and surgically removed tumor specimens were immunostained with the anti-CD44v9 specific antibodies.

Results

The intrinsic expression levels of CD44v9 in the biopsy specimens did not correlate with the chemoradioselection and patient survival. However, in N-CRS patients, the CD44v9-positive group demonstrated significantly (P = 0.008) worse prognosis, than the CD44v9-negative group. Multivariate analyses demonstrated that among four candidate factors (T, N, response to CCRT, and CD44v9), CD44v9 positivity (HR: 3.145, 95% CI: 1.235–8.008, P = 0.0163) was significantly correlated with the poor prognosis, along with advanced N stage (HR: 3.525, 95% CI: 1.054–9.060, P = 0.0228). Furthermore, the survival rate of the CD44v9-induced group was significantly (P = 0.04) worse than the CD44v9-non-induced group.

Conclusions

CCRT-induced CD44v9-expressing CSCs appear to be a major hurdle to chemoradioselection. CD44v9-targeting seems to be a promising strategy to enhance the efficacy of chemoradioselection and consequent organ preservation and survival.     

Genetic Structure of Capelin (Mallotus villosus) in the Northwest Atlantic Ocean

Capelin (Mallotus villosus) is a commercially exploited, key forage-fish species found in the boreal waters of the North Pacific and North Atlantic Oceans. We examined the population structure of capelin throughout their range in the Canadian northwest Atlantic Ocean using genetic-based methods. Capelin collected at ten beach and five demersal spawning locations over the period 2002 through 2008 (N = 3,433 fish) were genotyped using six polymorphic microsatellite loci. Temporally distinct samples were identified at three beach spawning locations: Chance Cove, Little Lawn and Straitsview, Newfoundland. Four capelin stocks are assumed for fisheries management in the northwest Atlantic Ocean based on meristics, morphometrics, tag returns, and seasonal distribution patterns. Our results suggested groupings that were somewhat different than the assumed structure, and indicate at least seven genetically defined populations arising from two ancestral populations. The spatial mosaic of capelin from each of the two basal cluster groups explains much of the observed geographic variability amongst neighbouring samples. The genetic-defined populations were resolved at Jost’s D _est ≥ 0.01 and were composed of fish collected 1) in the Gulf of St. Lawrence, 2) along the south and east coasts of Newfoundland, 3) along coastal northern Newfoundland and southern Labrador, 4) along coastal northern Labrador, 5) near the Saguenay River, and at two nearshore demersal spawning sites, 6) one at Grebes Nest off Bellevue Beach on the east coast of Newfoundland, and 7) one off the coast of Labrador at Domino Run. Moreover, the offshore demersal spawners on the Scotian Shelf and Southeast Shoal appeared to be related to the inshore demersal spawners at Grebes Nest and in Domino Run and to beach spawners from the Gulf of St. Lawrence.