Murine tumor cells transduced with the gene for tumor necrosis factor-alpha. Evidence for paracrine immune effects of tumor necrosis factor against tumors

Studies of the anti-tumor activity of TNF-alpha in vivo have been hampered by the need to administer systemically toxic doses of the cytokine to obtain a curative response. To facilitate studies of the effect of high local concentrations of TNF-alpha on tumor growth and host immunity, a newly induced murine sarcoma was transduced with the gene for human TNF-alpha and the biologic characteristics of these cells were examined. We identified high and low TNF-producing tumor clones which exhibited stable TNF secretion over time. Significant amounts of membrane associated TNF were found in a high-TNF producing clone as well. No difference in the in vitro growth rates between TNF-producing and nonproducing cell lines was observed. In contrast, in vivo studies demonstrate that although unmodified parental tumor cells grew progressively when implanted s.c. in animals, tumor cells transduced with the TNF gene were found to regress in a significant number of animals after an initial phase of growth. This effect correlated with the amount of TNF produced and could be blocked with a specific anti-TNF antibody. Regressions of TNF-producing cells occurred in the absence of any demonstrable toxicity in the animals bearing these tumors. TNF-producing tumor cells could function in a paracrine fashion by inhibiting the growth of unmodified, parental tumor cells implanted at the same site. The ability of tumor cells to regress was abrogated by in vivo depletion of CD4+ or CD8+ T cell subsets and animals that had experienced regression of TNF-producing tumors rejected subsequent challenges of parental tumor. Our studies thus show that tumor cells elaborating high local concentrations of TNF regress in the absence of toxicity in the host and that this process requires the existence of intact host immunity. Studies of the lymphocytes infiltrating the gene modified tumors and attempts to use TNF gene modified tumor infiltrating lymphocytes to deliver high local concentrations of TNF to the tumor site without inducing systemic toxicity are underway.     

Developmental origins and homologies of the hyracoid dentition

Understanding the origins of morphological specializations in mammals is a key goal in evolutionary biology. It can be accomplished by studying dental homology, which is at the core of most evolutionary and developmental studies. Here, we focused on the evolution and development of the specialized dentition of hyraxes for which dental homologies have long been debated, and could have implications on early placental evolution. Specifically, we analysed dental mineralization sequences of the three living genera of hyraxes and 17 fossil species using X‐ray computed microtomography. Our results point out the labile position of vestigial upper teeth on jaw bones in extant species, associated with the frequently unusual premolar shape of deciduous canines over 50 Ma of hyracoid evolution. We proposed two evolutionary and developmental hypotheses to explain these original hyracoid dental characteristics. (a) The presence of a vestigial teeth on the maxilla in front of a complex deciduous canine could be interpreted as extra‐teeth reminiscent of early placental evolution or sirenians, an order phylogenetically close to hyracoids and showing five premolars. (b) These vestigial teeth could also correspond to third incisors with a position unusually shifted on the maxilla, which could be explained by the dual developmental origin of these most posterior incisors and their degenerated condition. This integrative study allows discussion on the current evolutionary and developmental paradigms associated with the mammalian dentition. It also highlights the importance of nonmodel species to understand dental homologies.     

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

Polymorphism, divergence, and the role of recombination in Saccharomyces cerevisiae genome evolution

A contentious issue in molecular evolution and population genetics concerns the roles of recombination as a facilitator of natural selection and as a potential source of mutational input into genomes. The budding yeast Saccharomyces cerevisiae, in particular, has injected both insights and confusion into this topic, as an early system subject to genomic analysis with subsequent conflicting reports. Here, we revisit the role of recombination in mutation and selection with recent genome-wide maps of population polymorphism and recombination for S. cerevisiae. We confirm that recombination-associated mutation does not leave a genomic signature in yeast and conclude that a previously observed, enigmatic, negative recombination-divergence correlation is largely a consequence of weak selection and other genomic covariates. We also corroborate the presence of biased gene conversion from patterns of polymorphism. Moreover, we identify significant positive relations between recombination and population polymorphism at putatively neutrally evolving sites, independent of other factors and the genomic scale of interrogation. We conclude that widespread natural selection across the yeast genome has left its imprint on segregating genetic variation, but that this signature is much weaker than in Drosophila and Caenorhabditis.     

Hyperglycemia, hypoxia and their combination exert oxidative stress and changes in antioxidant gene expression: studies on cultured rat embryos

INTRODUCTION: Hyperglycemia and hypoxia are well‐known teratogens that may affect many animal species, including man. We hypothesize that a combination of hypoxia and hyperglycemia will increase embryonic damage produced by either factor individually. We investigated the interrelationship between hyperglycemia and hypoxia and their effects on genes involved in the balance of embryonic redox status. METHODS: Rat embryos (10.5‐day‐old) were cultured for 28 hr in culture medium with about 6 mg/ml of glucose and 20% oxygen (hyperglycemia), with 10% oxygen (hypoxia) and 2.4 g/ml glucose (normal) or a combination of both 6 mg/ml glucose and 10% oxygen. Antioxidant capacity was determined by activity and gene expression of antioxidant enzymes: Cu/Zn SOD, Mn‐SOD, CAT, and GSH‐px using real time PCR. RESULTS: Hyperglycemia, hypoxia, or their combination, decreased embryonic growth and induced a high rate (62–78%) of anomalies mainly of the nervous system, heart, and limbs. CAT mRNA and GSH‐px mRNA were decreased in the malformed embryos exposed to hyperglycemia, to hypoxia or their combination. CAT mRNA was also decreased in the nonmalformed embryos subjected to hyperglycemia and hypoxia. Cu/Zn SOD mRNA was increased in all experimental embryos whether malformed or not, whereas Mn‐SOD was drastically decreased. Total SOD and CAT like activity were changed very little in the experimental embryos compared to controls. CONCLUSIONS: Both hyperglycemia, hypoxia, and their combination reduce embryonic growth and development, induce embryonic anomalies, and modify the expression of the principle antioxidant genes. However, hypoxia does not seem to enhance the damaging effects of hyperglycemia except its effects of embryonic growth. Birth Defects Res (Part B) 92:231–239, 2011. © 2011 Wiley‐Liss, Inc.     

ESR Detection of 1O2 Reveals Enhanced Redox Activity in Illuminated Cell Cultures

Low-energy visible light (LEVL) has previously been found to modulate various processes in different biological systems. One explanation for the stimulatory effect of LEVL is light-induced reactive oxygen species formation. In the present study, both sperm and skin cells were illuminated with LEVL and were found to generate singlet oxygen (¹O₂). The detection of ¹O₂ was performed using a trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy. In addition, we have shown that, together with ¹O₂ generation, LEVL illumination increases the reductive capacity of the cells, which explains the difficulties encountered in ¹O₂ detection. The potential of visible light to change the cellular redox state may explain the recently observed biostimulative effects exerted by LEVL.