Overexpression of the disease resistance gene Pto in tomato induces gene expression changes similar to immune responses in human and fruitfly

The Pto gene encodes a serine/threonine protein kinase that confers resistance in tomato (Lycopersicon esculentum) to Pseudomonas syringae pv tomato strains that express the type III effector protein AvrPto. Constitutive overexpression of Pto in tomato, in the absence of AvrPto, activates defense responses and confers resistance to several diverse bacterial and fungal plant pathogens. We have used a series of gene discovery and expression profiling methods to examine the effect of Pto overexpression in tomato leaves. Analysis of the tomato expressed sequence tag database and suppression subtractive hybridization identified 600 genes that were potentially differentially expressed in Pto-overexpressing tomato plants compared with a sibling line lacking Pto. By using cDNA microarrays, we verified changes in expression of many of these genes at various time points after inoculation with P. syringae pv tomato (avrPto) of the resistant Pto-overexpressing line and the susceptible sibling line. The combination of these three approaches led to the identification of 223 POR (Pto overexpression responsive) genes. Strikingly, 40% of the genes induced in the Pto-overexpressing plants previously have been shown to be differentially expressed during the human (Homo sapiens) and/or fruitfly (Drosophila melanogaster) immune responses.     

Dominant cataracts result from incongruous mixing of wild-type lens connexins

Gap junctions are composed of proteins called connexins (Cx) and facilitate both ionic and biochemical modes of intercellular communication. In the lens, Cx46 and Cx50 provide the gap junctional coupling needed for homeostasis and growth. In mice, deletion of Cx46 produced severe cataracts, whereas knockout of Cx50 resulted in significantly reduced lens growth and milder cataracts. Genetic replacement of Cx50 with Cx46 by knockin rescued clarity but not growth. By mating knockin and knockout mice, we show that heterozygous replacement of Cx50 with Cx46 rescued growth but produced dominant cataracts that resulted from disruption of lens fiber morphology and crystallin precipitation. Impedance measurements revealed normal levels of ionic gap junctional coupling, whereas the passage of fluorescent dyes that mimic biochemical coupling was altered in heterozygous knockin lenses. In addition, double heterozygous knockout lenses retained normal growth and clarity, whereas knockover lenses, where native Cx46 was deleted and homozygously knocked into the Cx50 locus, displayed significantly deficient growth but maintained clarity. Together, these findings suggest that unique biochemical modes of gap junctional communication influence lens clarity and lens growth, and this biochemical coupling is modulated by the connexin composition of the gap junction channels.     

Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation

Cell-extracellular matrix adhesion is an important determinant of cell morphology. We show here that migfilin, a LIM-containing protein, localizes to cell-matrix adhesions, associates with actin filaments, and is essential for cell shape modulation. Migfilin interacts with the cell-matrix adhesion protein Mig-2 (mitogen inducible gene-2), a mammalian homolog of UNC-112, and the actin binding protein filamin through its C- and N-terminal domains, respectively. Loss of Mig-2 or migfilin impairs cell shape modulation. Mig-2 recruits migfilin to cell-matrix adhesions, while the interaction with filamin mediates the association of migfilin with actin filaments. Migfilin therefore functions as an important scaffold at cell-matrix adhesions. Together, Mig-2, migfilin and filamin define a connection between cell matrix adhesions and the actin cytoskeleton and participate in the orchestration of actin assembly and cell shape modulation.     

Effects of resveratrol on the G(0)-G(1) transition and cell cycle progression of mitogenically stimulated human lymphocytes

Resveratrol (RSV) has been suggested to have cancer preventive properties, on the basis that it suppresses proliferation and induces apoptosis in various tumor cells. Here we test its cytostatic effects on peripheral blood human lymphocytes. RSV (up to 50 microM) had no detectable effects on resting lymphocytes. With the mitogen phytohemagglutin (PHA), however, RSV elicited concentration- and time-dependent responses in lymphocytes. RSV (>/=50 microM) prevented cell entry into the cell cycle, resulting in 99% suppression at 100 microM. The arrested lymphocytes following 24h treatment with 50 microM RSV had minimal RNA content, the feature characteristic of G(0) cells, and were blocked at the stage past the induction of cyclins D2 and D3 and prior to induction of cyclin E. Prolonged treatment (72h) of PHA-stimulated lymphocytes with 100 microM RSV showed a pronounced decrease in the expression of pRb, cyclins E and B, and reduction in p34cdc2 and PCNA. The activation-induced apoptosis was also reduced in the presence of >/=50 microM RSV. These data suggest that studies designed to test RSV efficacy as a chemopreventive agent should include evaluation of its immunomodulatory effect revealed by suppression of lymphocyte stimulation as well as its effect on apoptosis of stimulated lymphocytes.     

Changes in ceramide levels upon catechins-induced apoptosis in LoVo cells

It has been demonstrated that there is difference in the induction of apoptosis in LoVo cells by (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin (EC). In this study, we explored changes in ceramide levels upon the three catechins-induced apoptosis in LoVo cells. Addition of C2- and C6-ceramide to LoVo cells mimicked EGCG or EGC in leading to apoptotic death. Further measurement of intracellular ceramide content showed that the treatment of LoVo cells with EGCG or EGC resulted in a rapidly transient increase in ceramide content, and then back gradually to base line level, whereas the action of EC was just opposite to that of EGCG or EGC. These results suggest there is difference in the generation of intracellular ceramide by the three catechins and ceramide may take part in the regulation of EGCG- or EGC-induced apoptosis in LoVo cells.     

Dose-dependent biphasic activity of tRNA synthetase-associating factor,p43, in angiogenesis

Mammalian aminoacyl tRNA synthetases form a macromolecular protein complex with three non-enzymatic cofactors. Among these factors, p43 is also secreted to work as a cytokine on endothelial as well as immune cells. Here we investigated the activity of p43 in angiogenesis and determined the related mediators. It promoted the migration of endothelial cells at low dose but induced their apoptosis at high dose. p43 at low concentration activated extracellular signal-regulating kinase, which resulted in the induction and activation of matrix metalloproteinase 9. In contrast, p43 at high concentration activated Jun N-terminal kinase, which mediated apoptosis of endothelial cells. These results suggest that p43 is a novel cytokine playing a dose-dependent biphasic role in angiogenesis.     

GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms

We have identified and cloned GLUT14, a novel member of the glucose transporter family. GLUT14 (SLC2A14) maps to chromosome 12p13.3 (17.1M), about 10 Mb upstream of GLUT3, with which it shares remarkable identity. Until now GLUT14 was thought to be a pseudogene. It consists of 11 exons with a genomic organization similar to that of GLUT3 and likely resulted from a duplication of GLUT3. GLUT14 has two alternatively spliced forms; the shorter form of GLUT14 (GLUT14-S) consists of 10 exons and produces a 497-amino-acid protein that is 94.5% identical to GLUT3. The long form (GLUT14-L) has an additional exon and codes for a protein with 520 amino acids that differs from GLUT14-S only at the N-terminus. GLUT14-S/L contain 12 putative membrane-spanning helices along with sugar-transporter signature motifs that have previously been shown to be essential for sugar transport activity. The putative glycosylation sites of GLUT14-S/L are present in loop 1. In contrast to the expression of GLUT3 in many tissues, both isoforms of GLUT14 are specifically expressed in testis. The mRNA level of GLUT14 in testis is about four times higher than that of GLUT3. Interestingly, the ortholog of GLUT14 is not found in mice. The multiple duplications of GLUT genes suggest that the GLUT family probably emerged by gene duplications and mutations during evolution in different lineages.     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2–3 h, was significantly lower than that of the 3–6 h groups (31.0%), while not significantly different among 3–4 h (P < 0.05), 4–5 h, and 5–6 h groups (P≥0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.     

11H-Isoquino[4,3-c]cinnolin-12-ones; novel anticancer agents with potent topoisomerase I-targeting activity and cytotoxicity

Recent studies have identified 2,3-dimethoxy-8,9-methylenedioxy-11-[(2-dimethylamino)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1a) as a novel topoisomerase I-targeting agent with potent cytotoxic activity. The effect of varied substituents at the 11-position of 2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-ones on topoisomerase I-targeting activity and cytotoxicity was evaluated. Potent TOP1-targeting activity was observed when the 11-position was substituted with either a 2-(N,N-dimethylamino)ethyl, a 2-(N,N-diethylamino)ethyl, a n-butyl, or a 2-(pyrrolidin-1-yl)ethyl group. The addition of a beta-methyl group to 1a provided an analogue with dramatically reduced TOP1-targeting activity and cytotoxicity. Analogues of 1a wherein the 2-(N,N-dimethylamino)ethyl group was replaced with a (2-tetrahydrofuranyl)methyl, a 2-(piperidin-1-yl)ethyl, or a 2-(4-methylpiperazin-1-yl)ethyl substituent exhibited decreased activity as TOP1-targeting agents. Replacement of the dimethoxy groups of 1a with hydrogen atoms resulted in an analogue with significantly decreased TOP1-targeting activity and cytotoxicity. Removal of both the vicinal dimethoxyl groups and the methylenedioxy moiety resulted in a complete loss of TOP1-targeting activity. The presence of a 9-nitro substituent in place of the 8,9-methylenedioxy group of 1a resulted in a decrease in relative TOP1-targeting activity and cytotoxicity. Compounds 1a and the 11-n-butyl analogue 1d were evaluated for antitumor activity in the human tumor xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line MDA-MB-435 was used in these assays. At dose levels that approached its maximum tolerated dose, 1a proved to be effective in inhibiting tumor growth in vivo when administered orally or by ip injection.     

Prevention of in-stent restenosis: towards an in situ treatment?

The use of intracoronary stents represent a major breakthrough in the armamentarium of interventional cardiology. Stents reduce significantly the incidence of recurrent stenosis (in-stent restenosis) via an improved post-procedure luminal diameter and an abrogation of the constrictive remodeling of the arterial wall. However, stent-related arterial injury results in intense proliferative and inflammatory responses and severe intimal hyperplasia, which, in 20% to 40% of the patients, may end up with clinically significant in-stent restenosis. Efficient prevention of in-stent restenosis has yet to be found. Systemic treatments have failed because they don't take into account the specific physiopathology and, most importantly, the focal nature of in-stent intimal hyperplasia. Hence, local prevention appears to be a straightforward approach to the unsolved issue of in-stent restenosis. In situ beta- or gamma-irradiation (brachytherapy) has received much attention as a curative treatment of in-stent restenosis but is not indicated for prevention. In contrast, drug-releasing stents have been tested in experimental models and have already provided very promising results in randomized clinical trials. Most of clinical studies have been performed with the antiproliferative agents sirolimus and paclitaxel, but other agents are under scrutiny. In addition, important research is carried out, in which the efficacy of antiproliferative genes is investigated. Clearly, drug-releasing stents are on the verge of profoundly modifying our practice of interventional cardiology. However, several questions remain unanswered as regard to the long term efficacy/toxicity and the cost-effectiveness of this new approach.