Apoptosis and catastrophic cell death in benzo[a]pyrene-transformed human breast epithelial cells

Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzo[a]pyrene (BP) (BP1, BP1-E and BP1-E1 cells) and in BP1 cells transfected with the c-Ha-ras oncogene (BP1-Tras cells). Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line.     

Mapping of the Na+, K(+)-ATPase subunit alpha 2 (ATP1A2) and muscle phosphofructokinase (PFKM) genes in pig by somatic cell hybrid analysis

Two expressed sequence tags were isolated from a porcine skeletal muscle cDNA library and identified as the putative partial cDNAs of the porcine Na+, K(+)-ATPase subunit alpha 2 (ATP1A2) and muscle phosphofructokinase (PFKM) genes after sequencing and homology search. Results of analysis of a pig-rodent somatic cell hybrid panel by PCR allowed the assignments of ATP1A2 to porcine chromosome (chr) 4 and of PFKM to porcine chr 5. These assignments support previously observed conservation of syntenic relationships between human chr 1 and porcine chr 4 and between human chr 12 and porcine chr 5.     

Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion

Ischemia-reperfusion injury is a microvascular event documented in numerous in vivo animal models. In animal models, prostaglandin and prostaglandin analogues have been found to ameliorate reperfusion injury. These studies were undertaken to evaluate human microvascular endothelial PGE(1) synthesis during in vitro ischemia followed by reperfusion. Human (neonatal) microvascular endothelial cell (MEC) cultures (n = 6) were subjected to sequential 2 h periods of normoxia (20% O(2)), ischemia (1.5% O(2)), and reperfusion (20% O(2)). Prostaglandin E(2) synthesis in conditioned media was determined by ELISA. Steady state levels of MEC prostaglandin H synthase (PGHS)-1 and -2 mRNA were assessed at the end of each 2-h period using RT-PCR and a quantitative mRNA ELISA. MEC PGHS protein levels were analyzed using an ELISA. PGE(1) release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE(1) release returned to normoxic levels at 30, 60, and 90 min, and exceeded normoxic levels at 120 min. PGHS-1 mRNA levels were undetectable during all experimental conditions. PGHS-2 mRNA levels were unchanged by ischemia, but were decreased by reperfusion. In contrast, PGHS-2 protein levels increased 3-fold during ischemia, and remained elevated during reperfusion. Human MEC do not express PGHS-1 mRNA in vitro. Prolonged ischemia decreases MEC PGE(1) synthesis, and stimulates increased PGHS-2 protein levels without altering the steady state levels of COX-2 mRNA. During reperfusion, increased PGHS-2 protein levels persist and are associated with stimulated PGE(2) secretion, despite relative decreases in PGHS-2 mRNA.     

Evidence for the notch signaling pathway on the role of estrogen in angiogenesis

We have investigated the molecular mechanisms involved in 17 beta-estradiol-induced angiogenic pathway. We show here that 17 beta-estradiol promoted a 6-fold increase in Jagged1 expression and an 8-fold increase in Notch1 expression by cDNA arrays in breast cancer MCF7 cells. Interestingly, Jagged1 was abrogated by incubation with the estrogen antagonist, ICI182,780. A similar up-regulation of both Notch1 receptor and Jagged1 ligand was found in endothelial cells. Additionally, imperfect estrogen-responsive elements were found in the 5' untranslated region of Notch1 and Jagged1 genes. Treatment with 17 beta-estradiol also led to an activation of Notch signaling in MCF7 cells expressing Notch1 reporter gene or by promoting Jagged1-induced Notch signaling in coculture assays. Inoculation of MCF7 cells in 17 beta-estradiol-treated nude mice resulted in up-regulation of Notch1 expression as well as increased number of tumor microvessels in comparison to placebo-treated mice. Notch1-expressing endothelial cell cultures formed cord-like structures on Matrigel in contrast to cells expressing a dominant-negative form of Notch1, emphasizing the relevance of Notch1 pathway in vessel assembly. Finally, Notch1-expressing MCF7 cells up-regulated hypoxia-inducible factor 1 alpha gene, a well-known angiogenic factor that clustered with Notch1 gene. This study implicates Notch signaling in the cross talk between 17 beta-estradiol and angiogenesis.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury

Many older patients, because of their high prevalence of coronary artery disease, are candidates for percutaneous coronary interventions (PCI), but the effects of vascular aging on restenosis after PCI are not yet well understood. Balloon injury to the right carotid artery was performed in adult and old rats. Vascular smooth muscle cell (VSMC) proliferation, apoptotic cell death, together with Akt induction, telomerase activity, p27kip1, and endothelial nitric oxide synthase (eNOS) expression was assessed in isolated arteries. Neointima hyperplasia and vascular remodeling along with endothelial cell regeneration were also measured after balloon injury. Arteries isolated from old rats exhibited a significant reduction of VSMC proliferation and an increase in apoptotic death after balloon injury when compared with adult rats. In the vascular wall of adult rats, balloon dilation induced Akt phosphorylation, and this was barely present in old rats. In arteries from old rats, Akt-modulated cell cycle check points like telomerase activity and p27kip1 expression were decreased and increased, respectively, compared with adults. After balloon injury, old rats showed a significant reduction of neointima formation and an increased vascular negative remodeling compared with adults. These results were coupled by a marked delay in endothelial regeneration in aged rats, partially mediated by a decreased eNOS expression and phosphorylation. Interestingly, chronic administration of L-arginine prevented negative remodeling and improved reendothelialization after balloon injury in aged animals. A decreased neointimal proliferation, an impaired endothelial regeneration, and an increase in vascular remodeling after balloon injury were observed in aged animals. The molecular mechanisms underlying these responses seem to be a reduced Akt and eNOS activity.     

Redox Control of Signal Transduction,Gene Expression and Cellular Senescence

Reactive oxygen species (ROS) act as subcellular messengers in such complex cellular processes as mitogenic signal transduction, gene expression, regulation of cell proliferation, replicative senescence, and apoptosis. They serve to maintain cellular homeostasis and their production is under strict control. However, the mechanisms whereby ROS act are still obscure. Here we review recent advances in our understanding of signaling mechanisms and recent data about the involvement of ROS in: (i) the regulation of the mitogenic transduction elements, particularly protein kinases and phosphatases; (ii) the regulation of gene expression; and (iii) the induction of replicative senescence and the role, if any, in aging and age-related disorders.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

alpha-Glycerylphosphorylethanolamine rescues astrocytes from mitochondrial impairment and oxidative stress induced by amyloid beta-peptides

The present work shows that alpha-glycerylphosphorylethanolamine (alpha-GPE) is effective in recovering astrocytes from mitochondrial membrane integrity and potential derangement and cellular oxidative stress that occur under amyloid beta-peptides-induced reactive gliosis.alpha-Glycerylphosphorylethanolamine (alpha-GPE), a new compound with nootropic properties, known to improve in vivo the learning and memory processes, has been tested for its protective properties on an in vitro model of degeneration. Rat primary astrocytic cultures treated with two amyloid-derived peptides, Abeta((1-40)) and Abeta(3(pE)-42), showed a marked reduction of the mitochondrial redox activity and membrane potential, together with an increase of oxidative species production. Plasma membrane lipid peroxidation (LPO) as well as generation of peroxides is greatly increased under Abeta-peptides toxicity. These features, typical of the reactive gliosis that accompanies neuronal degeneration, were readily recovered by pretreatment with alpha-GPE. alpha-GPE, likely improving the fluidity of cell membrane, has the potential to recover astrocytes from the general redox derangement induced by different amyloid fragments and possibly to protect from inflammation, gliosis and neurodegeneration. This is the first evidence of an antioxidant effect of the ethanolamine derivative on a rat model of chronic gliosis.