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.     

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata)

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.     

Rectangular flap for horizontal upper vermilion tightness in secondary cleft lip deformity

Radical paring of the cleft edge during a primary cleft operation or repeated secondary surgeries can result in tightness of the upper lip. The degree of the resulting side-to-side tension can vary, from mild cases for which improvement is sought through realignment of the misplaced oral sphincter muscle in secondary revision, to severe cases for which the possibility of a lip switch flap must be considered. When the lip tightness accompanies more than three-quarters loss of the Cupid's bow, an Abbé flap is an alternative. However, the lip switch flap is far from ideal, in both artistic and functional perspectives, and should be avoided if at all possible in mild to moderate degrees of lip tightness. This study presents a method of correcting horizontal cleft upper lip tightness, especially of the vermilion. The method involves local transfer of an inferiorly based rectangular flap from the relatively redundant upper two-thirds to the lower one-third of the upper lip and vermilion. Primary indications for the technique include vermilion tightness with half to three-quarters loss of Cupid's bow. The method has the advantage of supplementing the horizontal lip dimension on the cleft side and restoring a natural Cupid's bow, thereby repositioning the shifted philtral column and adding fullness to the lower one-third of the upper lip. Incorporation of the upper lip scar in the rectangular flap removes ugly scars and spares the lower lip from surgical violation. The orbicularis sphincter function, as seen in facial animation, was well regained. Twenty unilateral and three bilateral cases with a maximal follow-up period of 4.5 years are presented.     

Phospholipase C-delta1 rescues intracellular Ca2+ overload in ischemic heart and hypoxic neonatal cardiomyocytes

Ischemia and simulated ischemic conditions cause intracellular Ca2+ overload in the myocardium. The relationship between ischemia injury and Ca2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-delta1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-gamma1 (6.8 ng/mg) and PLC-beta1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-delta1, but not PLC-beta1 and PLC-gamma1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-delta1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-delta1 by the calcium sensitive proteases may play important roles in intracellular Ca2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia.     

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.     

Birth of rats following nuclear exchange at the 2-cell stage

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.     

Total ear reconstruction in the devascularized temporoparietal region: I. Use of the contralateral temporoparietal fascial free flap

Total ear reconstruction by the use of contralateral temporoparietal fascial free flap and autogenous costal cartilage was performed in 16 patients presenting with a devascularized temporoparietal region resulting from trauma or prior surgery. The microsurgical success rate was 87.5 percent (14 of 16 transplants). On evaluation of the final aesthetic result in 11 patients followed up for more than 3 years, nine patients were graded good-to-excellent and two patients exhibited fair-to-poor results. Despite the relatively long operating hours and the comparatively low microsurgical success rate, ear reconstruction by autogenous tissue transplantation has proved to be an encouraging and worthwhile experience. This article presents the clinical cases and discusses the technical details.     

Stage-sensitive blockade of pituitary somatomammotrope development by targeted expression of a dominant negative epidermal growth factor receptor in transgenic mice

The epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-alpha (TGF alpha) are expressed in the anterior pituitary, and overexpression of TGF alpha in the lactotrope cells of the pituitary gland in transgenic mice results in lactotrope hyperplasia and adenomata, suggesting a role for EGFR signaling in pituitary cell proliferation. To address the role of EGFR signaling in pituitary development in vivo, we blocked EGFR signaling in transgenic mice using the dominant negative properties of a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr). We directed EGFR-tr expression to GH- and PRL- producing cells using GH and PRL promoters, and a tetracycline-inducible gene expression system, to allow temporal control of gene expression. EGFR-tr overexpression in GH-producing cells during embryogenesis resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and lactotrope development were blocked. However, when EGFR-tr overexpression was delayed to the postnatal period either by directing its expression with the PRL promoter or by delaying the onset of induction with tetracycline in the GH cells, no specific phenotype was observed. Lactotrope hyperplasia during pregnancy also occurred normally in the PRL-EGFR-tr mice. These data suggest that EGFR signaling is required for the differentiation and/or maintenance of somatomammotropes early in pituitary organogenesis but not later in life. (Molecular Endocrinology 15: 600-613, 2001)     

The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions

Mammalian homologues of the Drosophila polarity proteins Stardust, Discs Lost, and Crumbs have been identified as Pals1, Pals1-associated tight junction protein (PATJ), and human Crumbs homologue 1 (CRB1), respectively. We have previously demonstrated that PATJ, Pals1, and CRB1 can form a tripartite tight junction complex in epithelial cells and that PATJ recruits Pals1 to tight junctions. Here, we observed that the Pals1/PATJ interaction was not crucial for the ultimate targeting of PATJ itself to tight junctions. This prompted us to examine if any of the 10 post-synaptic density-95/Discs Large/zona occludens-1 (PDZ) domains of PATJ could bind to the carboxyl termini of known tight junction constituents. We found that the 6th and 8th PDZ domains of PATJ can interact with the carboxyl termini of zona occludens-3 (ZO-3) and claudin 1, respectively. PATJ missing the 6th PDZ domain was found to mislocalize away from cell contacts. Surprisingly, deleting the 8th PDZ domain had little effect on PATJ localization. Finally, reciprocal co-immunoprecipitation experiments revealed that full-length ZO-3 can associate with PATJ. Hence, the PATJ/ZO-3 interaction is likely important for recruiting PATJ and its associated proteins to tight junctions.