Regulation of tumor-associated macrophage (TAM) differentiation by NDRG2 expression in breast cancer cells

Macrophages are a major cellular component of innate immunity and are mainly known to have phagocytic activity. In the tumor microenvironment (TME), they can be differentiated into tumor-associated macrophages (TAMs). As the most abundant immune cells in the TME, TAMs promote tumor progression by enhancing angiogenesis, suppressing T cells and increasing immunosuppressive cytokine production. N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor gene, whose expression is down-regulated in various cancers. However, the effect of NDRG2 on the differentiation of macrophages into TAMs in breast cancer remains elusive. In this study, we investigated the effect of NDRG2 expression in breast cancer cells on the differentiation of macrophages into TAMs. Compared to tumor cell-conditioned medium (TCCM) from 4T1-mock cells, TCCM from NDRG2-overexpressing 4T1 mouse breast cancer cells did not significantly change the morphology of RAW 264.7 cells. However, TCCM from 4T1-NDRG2 cells reduced the mRNA levels of TAM-related genes, including MR1, IL-10, ARG1 and iNOS, in RAW 264.7 cells. In addition, TCCM from 4T1-NDRG2 cells reduced the expression of TAM-related surface markers, such as CD206, in peritoneal macrophages (PEM). The mRNA expression of TAM-related genes, including IL-10, YM1, FIZZ1, MR1, ARG1 and iNOS, was also downregulated by TCCM from 4T1-NDRG2 cells. Remarkably, TCCM from 4T1-NDRG2 cells reduced the expression of PD-L1 and Fra-1 as well as the production of GM-CSF, IL-10 and ROS, leading to the attenuation of T cell-inhibitory activity of PEM. These data showed that compared with TCCM from 4T1-mock cells, TCCM from 4T1-NDRG2 cells suppressed the TAM differentiation and activation. Collectively, these results suggest that NDRG2 expression in breast cancer may reduce the differentiation of macrophages into TAMs in the TME.     

Fecal microbiota and diets of muskox female adults and calves

In mammals, the gut microbiome is vertically transmitted during maternal lactation at birth. In this study, we investigated the gut microbiome and diets of muskox, a large herbivore inhabiting in the high Arctic. We compared the microbiota composition using bacterial 16S rRNA gene sequencing and diets using stable isotope analysis of muskox feces of six female adults and four calves on Ella Island, East Greenland. Firmicutes were the most abundant bacterial phylum in both the adults and calves, comprising 94.36% and 94.03%, respectively. Significant differences were observed in the relative abundance of the two Firmicutes families. The adults were primarily dominated by Ruminococcaceae (73.90%), and the calves were dominated by both Ruminococcaceae (56.25%) and Lachnospiraceae (24.00%). Stable isotope analysis of the feces in the study area revealed that both adults and calves had similar ranges of ¹³C and ¹⁵N, likely derived from the dominant diet plants. Despite their similar diets, the different gut microbiome compositions in muskox adults and calves indicate that the gut microbiome of the calves may not be fully colonized to the extent of that of the adults.     

Development of a novel expression vector system using Spodoptera exigua nucleopolyhedrovirus

To develop a novel Spodoptera exigua nucleopolyhedrovirus (SeNPV) expression vector system, we examined characteristics of the SeNPV polyhedrin expression in S. exigua cells (Se301). While the extracellular virus titer of SeNPV was 100-fold lower than that of Autographa californica nucleopolyhedrovirus (AcNPV), the levels of polyhedral inclusion body (PIB) formation and polyhedrin expression were higher in SeNPV. To investigate foreign gene expression under the control of the polyhedrin promoter, polyhedrin-based transfer vector pSeKSK2 was constructed, and then recombinant virus SeK1-LacZ was constructed by inserting E. coli lacZ gene as a reporter gene into a genomic DNA of SeNPV using this transfer vector. The beta-Galactosidase activity of SeK1-LacZ in Se301 was about 1.3 times higher than that of BacPAK6. Thus, the SeNPV expression vector system constructed in this study would be very useful in the expression of foreign proteins, specifically for the enhancement of the pesticidal properties of SeNPV by inserting pesticidal genes.     

Molecular cloning,expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H₂O₂ (100M) or Fe³⁺/O₂/DTT oxidation system, but not by ABA (50 M) or GA₃ (10 M). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (C2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The C2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The C2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the C2C-Prx exhibits peroxidase activity on H₂O₂.     

Role of ERK1/2 in uterine contractility and preterm labor in rats

The present study tested the hypothesis that ERK activation is an essential step in the onset of labor in a rat model of preterm labor. The administration of RU-486, an antiprogesterone agent, to rats induced preterm delivery 22.2 +/- 0.24 h after treatment. Changes in basal signaling events were studied in myometrial tissue from CO(2)-euthanized rats. Rats treated with RU-486 displayed a dramatically increased in vitro uterine contractility compared with gestational stage-matched, sham-treated rats. In vitro contractility was not significantly different from that during spontaneous labor. During RU-486-induced preterm labor, as previously described for spontaneous labor, ERK phosphorylation levels increased, as did phosphorylation of caldesmon at Ser(789), an ERK phosphorylation site. Also, a small but significant increase in 20-kDa myosin light chain phosphorylation was seen at a constant intracellular pCa of 7. When rats were chronically treated with an agent that prevents ERK activation, U-0126, the onset of RU-486-induced preterm labor was delayed in a statistically significant manner. Chronic in vivo treatment with U-0126 also significantly inhibited the RU-486-induced increase in in vitro contractility and ERK and caldesmon phosphorylation but did not alter the RU-486-induced increase in 20-kDa myosin light chain phosphorylation. These data indicate that ERK activation is a component of the multiple events leading to the development of labor in this rat model. We suggest that the ERK pathway could possibly be used to identify targets for the development of a novel class of tocolytic agents.     

Isolation and Characterization of a Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">morrisoni</Emphasis> PG-14 Encoding δ-Endotoxin Cry1Ac

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

p57KIP2 modulates stress-activated signaling by inhibiting c-Jun NH2-terminal kinase/stress-activated protein Kinase

p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.