Mammary gland involution is a physiological process in which the entire organ is remodeled through the process of apoptosis. Apoptosis of secretory alveolar cells is initiated at the time of weaning, followed by the collapse and disappearance of the entire lobuloalveolar compartment. While apoptotic figures were rare in mammary epithelium of lactating mice, their number increased after weaning and reached a maximum on day 3 of involution. Active cell death continued until day 5 after weaning and only little parenchyma remained on day 8, when remodeling of the gland was completed. Bax mRNA levels increased during the first day of involution independent of the presence or absence of p53. Bax protein was detected in an increasing number of cells after weaning, peaking at day 3 and decreasing thereafter. Low levels of bcl-x mRNA and protein were present during lactation, followed by a sharp increase during the first 2 days of involution. The bcl-xS splice variant of bcl-x can promote cell death, and bcl-xL has a protective function in cell culture. The ratio of bcl-xS versus bcl-xL remained stable in the virgin, pregnant and lactating gland. However, during the first 2 days of involution, bcl-xS expression increased six-fold more than bcl-xL. To further evaluate the role of Bcl-xS which was less abundant in the mammary cells than Bcl-xL, cotransfection studies were performed in cell culture. They confirmed that Bcl-xS protein can facilitate apoptosis even when Bcl-xL is present in excess. These findings point to a significant role for Bax and Bcl-xS in the regulation of apoptosis of secretory alveolar cells during involution.

Atherogenic lipoproteins stimulate O2- formation and induction of apoptosis in HUVECs and RA, and may thereby influence the pathogenesis of atherosclerosis.

1 The physiological role of the angiotensin II AT2 receptor subtype is not fully characterized. We studied whether AT2 receptor could antagonize AT1 mediated superoxide formation in endothelial cells. 2 In quiescent human umbilical vein endothelial cells (HUVEC) superoxide formation was measured after long-term incubation (6 h) with angiotensin II in the presence or absence of its receptor blocker candesartan (AT1) or PD123319 (AT2) using the cytochrome c assay. In separate experiments, the e ects of AT2 mediated e ects on activities of cellular phosphates including the src homology 2 domain containing phosphatases (SHP-1) was studied. 3 The basal superoxide formation (0.19+0.03 nmol superoxide mg protein 71 min 71 ) in HUVEC was increased by 37.1% after exposure to angiotensin II (100 nM,) which was due to an activation of a NAD(P)H oxidase. This was abolished by candesartan (1 mM) as well as the tyrosine kinase inhibitor genistein. In contrast, blockade of AT2 receptors by PD123319 enhanced the superoxide formation by 73.7% in intact cells. Stimulation of AT2 went along with an increased activity of tyrosine phosphatases in total cell lysates (29.8%) and, in particular, a marked stimulation of src homology 2 domain containing phosphatases (SHP-1, by 293.4%). The tyrosine phosphatase inhibitor vanadate, in turn, prevented the AT2 mediated e ects on superoxide formation. The expression of both angiotensin II receptor subtypes AT1 and AT2 was con®rmed by RT ± PCR analysis. 4 It is concluded that AT2 functionally antagonizes the AT1 induced endothelial superoxide formation by a pathway involving tyrosine phosphatases.

No abstract

The postgenomic era is characterized by an almost intimidating amount of information regarding the sequences and expression of previously unknown genes. In response, researchers have developed an increasing interest in functional studies. At the start of such a study, one may have little more than sequence information and bioinformatic annotation. The next step is to hypothesize a potential role in the context of a cell. Testing of the hypothesis needs to be fast, cheap, and applicable to a large number of genes. Knockdown methods that rely on binding of antisense oligonucleotides to mRNA combined with a subsequent functional assay in cell culture fulfil these requirements: sequence information is sufficient for synthesis of active inhibitors. Depending on the in vitro model chosen, knockdown of gene expression can be achieved with medium or even high throughput. The two most popular methods of knockdown in cell culture are the use of antisense oligonucleotides that rely on ribonuclease H (RNAse H)-dependent cleavage of mRNA, and RNA interference triggered by small double-stranded RNA molecules. Both methods act in a sequence-specific manner and can give efficient knockdown. In both cases, researchers struggle with nonspecific "off-target" effects and the difficulty of site selection. Studies that compare the methods differ in their judgment as to which method is superior.

No abstract

Atherogenic lipoproteins, oxidative stress, and cell death. bits produce relatively high amounts of O 2 Ϫ [10], and Background. Glomerulosclerosis and atherosclerosis are oxygen radicals are also involved in the pathogenesis of chronic inflammatory processes that may be influenced by oxiglomerular diseases [11]. We therefore hypothesize that dized lipoproteins, oxidized low-density lipoproteins (oxLDL), a major contribution of oxidized lipoproteins to the deand oxidized lipoprotein(a) [oxLp(a)]. We hypothesize that velopment of glomerulosclerosis and atherosclerosis these lipoproteins contribute to the development of glomerulosclerosis and atherosclerosis through the induction of oxidative consists of the induction of oxidative stress. Oxidative stress, which influences cell viability. We therefore determined stress attenuates cell viability (apoptotic and necrotic the impact of oxLDL and oxLp(a) on O 2 Ϫ formation and on cell death) [12] and, in turn, may influence the developnecrotic and apoptotic cell death in vascular and glomerular ment of glomerulosclerosis [13] and atherosclerosis. Incells. deed, vascular regions that are prone to the development Methods. The impact of human LDL and Lp(a) (oxidized with CuSO 4 ) on O 2 Ϫ formation (detected with a chemiluminesof atherosclerotic lesions are characterized by increased cence method), apoptosis, and necrosis (determined with the cell turnover during the early phase of the disease [14]. annexin assay) was studied in cultured human umbilical vein Apoptosis has also been found in human and animal endothelial cells (ECs) and in cultured human mesangial cells atherosclerotic lesions [15, 16]. It was therefore the aim (MCs). of this study to determine the impact of oxidized low-Results. O 2 Ϫ formation was increased by 10 g/ml oxLDL (by factor 2.5 in ECs) and by 5 g/ml oxLp(a) (by factor 3.5 density lipoproteins (oxLDL), and oxidized lipoproin ECs). OxLDL and oxLp(a) both significantly and dosetein(a) [oxLp(a)] on O 2 Ϫ formation and on necrotic and dependently increased the rate of apoptotic cell death in ECs apoptotic cell death in vascular and glomerular cells. and in MCs, with oxLp(a) being the more potent stimulus that also caused necrosis. The induction of apoptosis by oxLDL and oxLp(a) in ECs and MCs was enhanced by inhibition METHODS of the endogenous superoxide dismutase (SOD) with diethyldithio-carbamate and was blunted by the antioxidants N-acetyl-Human LDL and Lp(a) were isolated by density gradicysteine, vitamin C ϩ E, SOD, and catalase, suggesting that ent ultracentrifugation from fresh human plasma and, in oxidative stress was the stimulus for apoptosis.case of Lp(a), by additional lysine-sepharose 4B chroma-Conclusions. These data suggest that oxLDL and oxLp(a)

We measured oxidative stress caused by lipoproteins in human umbilical vein endothelial cells and rabbit aorta. Oxidized lipoprotein(a) was the more potent stimulus over oxidized low density lipoprotein, and we believe that both influence the pathogenesis of atherosclerosis.