Disrupted retinal development in the embryonic belly spot and tail mutant mouse.
(9/11751)
The Belly spot and tail (Bst) semidominant mutation, mapped to mouse Chromosome 16, leads to developmental defects of the eye, skeleton, and coat pigmentation. In the eye, the mutant phenotype is characterized by the presence of retinal colobomas, a paucity of retinal ganglion cells, and axon misrouting. The severity of defects in the Bst/+ retina is variable among individuals and is often asymmetric. In order to determine the role of the Bst locus during retinal morphogenesis, we searched for the earliest observable defects in the developing eye. We examined the retinas of Bst/+ and +/+ littermates from embryonic day 9.5 (E9.5) through E13.5 and measured retinal size, cell density, cell death, mitotic index, and cell birth index. We have found that development of the Bst/+ retina is notably dilatory by as early as E10.5. The affected retinas are smaller than their wildtype counterparts, and optic fissure fusion is delayed. In the mutant, there is a marked lag in the exit of retinal cells from the mitotic cycle, even though there are no observable differences in the rate of cellular proliferation or cell death between the two groups. We hypothesize that Bst regulates retinal cell differentiation and that variability of structural defects in the mutant, such as those affecting optic fissure fusion, is a reflection of the extent of developmental delay brought about by the Bst mutation. (+info)
Glucocorticoids inhibit serum depletion-induced apoptosis in T lymphocytes expressing Bcl-2.
(10/11751)
Depletion of growth factors and glucocorticoids are known to induce apoptosis and inhibit growth in T lymphocytes. We have examined the effect of Bcl-2 expression on the cellular response to growth factor depletion in the presence or absence of glucocorticoids. Cell growth was determined by cell counting and viability was quantitated by dye exclusion. Apoptosis was evaluated by flow cytometry, analysis of DNA integrity, and enzymatic determination of caspase-3-like activity. Serum depletion and glucocorticoid administration inhibited cell growth and stimulated apoptosis in Bcl-2 negative cells. Cotreatment with both stimuli had additive effects on apoptosis but not on inhibition of cell growth. Bcl-2 expression abrogated the repressive effect of glucocorticoids on apoptosis but not on cell growth. In contrast, neither apoptosis nor growth inhibition induced by serum depletion of cells was blocked by Bcl-2 expression. However, glucocorticoid treatment of Bcl-2-overexpressing cells protected them from apoptosis induced by serum depletion. Glucocorticoid protection of Bcl-2-overexpressing cells from serum depletion-induced apoptosis was mimicked by other inducers of apoptosis, which act to inhibit protein synthesis. These data suggest that Bcl-2 expression can switch the effect of glucocorticoids from proapoptotic to antiapoptotic when lymphocytes expressing Bcl-2 are exposed to other apoptotic stimuli. (+info)
Bcl-2 alters the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins.
(11/11751)
Oxidized low density lipoproteins (oxLDL) participate in atherosclerosis plaque formation, rupture, and subsequent thrombosis. Because oxLDL are toxic to cultured cells and Bcl-2 protein prevents apoptosis, the present work aimed to study whether Bcl-2 may counterbalance the toxicity of oxLDL. Two experimental model systems were used in which Bcl-2 levels were modulated: 1) lymphocytes in which the (high) basal level of Bcl-2 was reduced by antisense oligonucleotides; 2) HL60 and HL60/B (transduced by Bcl-2) expressing low and high Bcl-2 levels, respectively. In cells expressing relatively high Bcl-2 levels (lymphocytes and HL60/B), oxLDL induced mainly primary necrosis. In cells expressing low Bcl-2 levels (antisense-treated lymphocytes, HL60 and ECV-304 endothelial cells), the rate of oxLDL-induced apoptosis was higher than that of primary necrosis. OxLDL evoked a sustained calcium rise, which is a common trigger to necrosis and apoptosis since both types of cell death were blocked by the calcium chelator EGTA. Conversely, a sustained calcium influx elicited by the calcium ionophore A23187 induced necrosis in cells expressing high Bcl-2 levels and apoptosis in cells expressing low Bcl-2 levels. This suggests that Bcl-2 acts downstream from the calcium peak and inhibits only the apoptotic pathway, not the necrosis pathway, thus explaining the apparent shift from oxLDL-induced apoptosis toward necrosis when Bcl-2 is overexpressed. (+info)
Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage.
(12/11751)
When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Therefore, annexin V-binding, in conjunction with flow cytometry, was used to evaluate the integrity of the sperm plasma membrane after different cryostorage protocols: i.e. 10% (v/v) glycerol; sperm maintenance medium (MM); freezing medium TEST yolk buffer (TYB); or cryostorage without protection (cryoshock). Using a combination of two fluorescent dyes, annexin V and propidium iodide (PI), led to three groups of spermatozoa being identified: (i) viable spermatozoa (annexin V-negative and PI-negative); (ii) dead spermatozoa (annexin V-positive and PI-positive); and (iii) cells with impaired but integer plasma membrane (annexin V-positive and PI-negative). The percentage of vital annexin V-negative spermatozoa increased significantly (P < 0.05) from spermatozoa treated by cryoshock (15.0+/-1.2%) to spermatozoa cryopreserved by TYB (26.6+/-2.2%) via cryopreservation by 10% (v/v) glycerol (19.9+/-1.6%) and by MM (22.2 1.8%) and was associated with the percentage of motile spermatozoa (17.6+/-3.4% by glycerol; 19.6+/-3.7% by MM and 22.6+/-3.9% by TYB; P = 0.0001). Of the spermatozoa, 12-22% were annexin V-positive even though they did not bind to PI, indicating viability before as well as after cryostorage. The percentage of vital annexin V-positive spermatozoa was significantly correlated with different sperm motility parameters (velocity straight linear, r = 0.601, P = 0.018; percentage of linearly motile spermatozoa: r = 0.549, P = 0.034). We, therefore, concluded that annexin V-binding is more sensitive in detecting a deterioration of membrane functions than PI staining, and that a considerable percentage of spermatozoa might have dysfunctional plasma membranes besides dead or moribund cells. Of the cryopreservation protocols tested, TYB yielded the most viable spermatozoa. Therefore, we advocate the use of the annexin V-binding assay for the evaluation of the quality and integrity of spermatozoa. (+info)
Specification of distinct dopaminergic neural pathways: roles of the Eph family receptor EphB1 and ligand ephrin-B2.
(13/11751)
Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens/olfactory tubercle, respectively, constituting mesostriatal and mesolimbic pathways. The molecular signals that confer target specificity of different dopaminergic neurons are not known. We now report that EphB1 and ephrin-B2, a receptor and ligand of the Eph family, are candidate guidance molecules for the development of these distinct pathways. EphB1 and ephrin-B2 are expressed in complementary patterns in the midbrain dopaminergic neurons and their targets, and the ligand specifically inhibits the growth of neurites and induces the cell loss of substantia nigra, but not ventral tegmental, dopaminergic neurons. These studies suggest that the ligand-receptor pair may contribute to the establishment of distinct neural pathways by selectively inhibiting the neurite outgrowth and cell survival of mistargeted neurons. In addition, we show that ephrin-B2 expression is upregulated by cocaine and amphetamine in adult mice, suggesting that ephrin-B2/EphB1 interaction may play a role in drug-induced plasticity in adults as well. (+info)
Continuing postischemic neuronal death in CA1: influence of ischemia duration and cytoprotective doses of NBQX and SNX-111 in rats.
(14/11751)
BACKGROUND AND PURPOSE: Transient forebrain ischemia results in a 24- to 72-hour delayed loss of CA1 neurons. Previous work has not assessed whether insult durations can vary the degree and maturation rate of CA1 injury and whether there are different ultrastructural features of death after brief or severe ischemia. We also tested whether known cytoprotective drugs achieve permanent or transient neuroprotection. METHODS: In the first experiment, ischemia was induced for 5, 15, or 30 minutes with the use of the 4-vessel occlusion rat model with 1- to 28-day survival. Others subjected to 5 or 15 minutes of ischemia and allowed to survive for 14 or 7 days, respectively, were examined with electron microscopy. Finally, we determined whether NBQX (30 mg/kg x3 at 0 or 6 hours after ischemia), an AMPA antagonist, and SNX-111 (5 mg/kg at 6 hours after ischemia), an N-type Ca2+ channel antagonist, provided enduring CA1 protection against 10 minutes of ischemia. RESULTS: CA1 damage was not detected at 24 hours. Thirty minutes of ischemia produced 47% and 84% CA1 damage at 2 and 3 days, respectively. A 15-minute occlusion yielded 11%, 74%, and 86% loss at 2, 3, and 7 days, respectively. Five minutes of ischemia produced an even slower progression with 24%, 52%, and 59% loss at 3, 7, and 14 days, respectively. Ultrastructural examination after 5 and 15 minutes of ischemia revealed necrosis with no morphological evidence of apoptosis. Both NBQX (P<0.021) and SNX-111 (P<0.001) significantly reduced CA1 death at 7 days (/=80%) compared with saline treatment ( approximately 79%). CONCLUSIONS: Brief forebrain ischemia results in a slower progression of CA1 loss than more severe insults. Nonetheless, neuronal injury had necrotic, not apoptotic, morphology. NBQX and SNX-111 only postponed CA1 injury. (+info)
Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death.
(15/11751)
Over recent years, it has become clear that mitochondria play a central role in many key aspects of animal physiology and pathophysiology. Their central and ubiquitous task is clearly the production of ATP. Nevertheless, they also play subtle roles in glucose homeostasis, acting as the sensor for substrate supply in the transduction pathway that promotes insulin secretion by the pancreatic -cell and that modulates the excitability of the hypothalamic glucose-sensitive neurons involved in appetite control. Mitochondria may also act as sensors of availability of oxygen, the other major mitochondrial substrate, in the regulation of respiration. Mitochondria take up calcium, and the high opacity mitochondrial calcium uptake pathway provides a mechanism that couples energy demand to increased ATP production through the calcium-dependent upregulation of mitochondrial enzyme activity. Mitochondrial calcium accumulation may also have a substantial impact on the spatiotemporal dynamics of cellular calcium signals, with subtle differences of detail in different cell types. Recent work has also revealed the centrality of mitochondrial dysfunction as an irreversible step in the pathway to both necrotic and apoptotic cell death. This review looks at recent developments in these rapidly evolving areas of cell physiology in an attempt to draw together disparate areas of research into a common theme. (+info)
Toward a leukemia treatment strategy based on the probability of stem cell death: an essay in honor of Dr. Emil J Freireich.
(16/11751)
Dr. Emil J Freireich is a pioneer in the rational treatment of cancer in general and leukemia in particular. This essay in his honor suggests that the cell kill concept of chemotherapy of acute myeloblastic leukemia be extended to include two additional ideas. The first concept is that leukemic blasts, like normal hemopoietic cells, are organized in hierarchies, headed by stem cells. In both normal and leukemic hemopoiesis, killing stem cells will destroy the system; furthermore, both normal and leukemic cells respond to regulators. It follows that acute myelogenous leukemia should be considered as a dependent neoplasm. The second concept is that cell/drug interaction should be considered as two phases. The first, or proximal phase, consists of the events that lead up to injury; the second, or distal phase, comprises the responses of the cell that contribute to either progression to apoptosis or recovery. Distal responses are described briefly. Regulated drug sensitivity is presented as an example of how distal responses might be used to improve treatment. (+info)