p53 deficiency fails to prevent increased programmed cell death in the Bcl-X(L)-deficient nervous system.

Bcl-X(L) mice display a similar neurodevelopmental phenotype as rb, DNA ligase IV, and XRCC4 mutant embryos, suggesting that endogenous Bcl-X(L) expression may protect immature neurons from death caused by DNA damage and/or cell cycle dysregulation. To test this hypothesis, we generated bcl-x/p53 double mutants and examined neuronal cell death in vivo and in vitro. Bcl-X(L)-deficient primary telencephalic neuron cultures were highly susceptible to the apoptotic effects of cytosine arabinoside (AraC), a known genotoxic agent. In contrast, neurons lacking p53, or both Bcl-X(L) and p53, were markedly, and equivalently, resistant to AraC-induced caspase-3 activation and death in vitro indicating that Bcl-X(L) lies downstream of p53 in DNA damage-induced neuronal death. Despite the ability of p53 deficiency to protect Bcl-X(L)-deficient neurons from DNA damage-induced apoptosis in vitro, p53 deficiency had no effect on the increased caspase-3 activation and neuronal cell death observed in the developing Bcl-X(L)-deficient nervous system. These findings suggest that Bcl-X(L) expression in the developing nervous system critically regulates neuronal responsiveness to an apoptotic stimulus other than inadequate DNA repair or cell cycle abnormalities.

Chloroquine-induced neuronal cell death is p53 and Bcl-2 family-dependent but caspase-independent.

Chloroquine is a lysosomotropic agent that causes marked changes in intracellular protein processing and trafficking and extensive autophagic vacuole formation. Chloroquine may be cytotoxic and has been used as a model of lysosomal-dependent cell death. Recent studies indicate that autophagic cell death may involve Bcl-2 family members and share some features with caspase-dependent apoptotic death. To determine the molecular pathway of chloroquine-induced neuronal cell death, we examined the effects of chloroquine on primary telencephalic neuronal cultures derived from mice with targeted gene disruptions in p53, and various caspase and bcl-2 family members. In wild-type neurons, chloroquine produced concentration- and time-dependent accumulation of autophagosomes, caspase-3 activation, and cell death. Cell death was inhibited by 3-methyladenine, an inhibitor of autophagic vacuole formation, but not by Boc-Asp-FMK (BAF), a broad caspase inhibitor. Targeted gene disruptions of p53 and bax inhibited and bcl-x potentiated chloroquine-induced neuron death. Caspase-9- and caspase-3-deficient neurons were not protected from chloroquine cytotoxicity. These studies indicate that chloroquine activates a regulated cell death pathway that partially overlaps with the apoptotic cascade.

Bax deficiency prevents the increased cell death of immature neurons in bcl-x-deficient mice.

The intracellular balance between pro- and antiapoptotic members of the Bcl-2 gene family is thought to regulate cell death. Targeted disruption of bcl-x, a death repressing member, causes massive cell death of immature neurons in the developing mouse CNS, whereas targeted disruption of bax, a proapoptotic member, blocks the death of specific populations of sympathetic and motor neurons. In the present study, mice deficient in both Bcl-xL and Bax (bcl-x-/-/bax-/-) are used to examine the relative significance and potential interactions of Bcl-xL and Bax during early CNS development. bcl-x-/-/bax-/- mice demonstrate greatly reduced levels of apoptosis both in vivo and in vitro compared with the CNS of Bcl-xL-deficient mice, as assessed by histology and terminal deoxytransferase-mediated deoxyuridine triphosphate nick end-labeling. Bax-deficient mice, however, contain occasional apoptotic cells in the developing CNS, and cultures of bax-deficient telencephalic cells demonstrate similar levels of apoptosis as wild-type cultures. These results suggest that Bax critically interacts with Bcl-xL to regulate survival of immature neurons, but indicate that other cell death regulating proteins, in addition to Bcl-xL and Bax, also function during CNS development.

Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.

To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.

The electroretinogram in preterm infants.

With the hope that the electroretinogram (ERG) in preterm infants could help clarify their vitamin A requirements, a technique for recording the full-field ERG in the neonate is described. One hundred seventy-seven ERGs were performed in 59 preterm and 52 term infants. An ERG was recorded as soon as 7 hours after birth and as early as 30 weeks after conception. In preterm infants the a-wave latency was longer and the amplitude less than in term infants of the same age. The amplitude of the ERG in preterm infants increased with the duration of light exposure. Longitudinal data on 15 preterm infants showed a reduction in a-wave latency. None of the ERG findings correlated with postconceptional age, which suggests that the duration of light exposure is a major determinant of the ERG pattern in preterm infants. Despite low circulating levels of retinol, no correlations with any of the ERG values were found.

Vitamin A status of preterm infants: correlation between plasma retinol concentration and retinol dose response.

Retinol dose response tests were performed on 83 preterm infants shortly before discharge by giving orally 5000 IU of an aqueous dispersion of retinol. Predose plasma retinol concentrations were 2.5-20.5 micrograms/dL (0.087-0.72 mumol/L) and the retinol dose responses were 0-59.8%. The regression of retinol dose response on predose retinol was -0.58. There was a parallel increase in both retinol and retinol-binding protein and an increase in the molar ratio of retinol-binding protein to prealbumin. Prealbumin did not increase. These findings suggest that preterm infants have reduced liver stores of vitamin A.

Vitamin A status of preterm infants: the influence of feeding and vitamin supplements.

Consecutive weekly determinations of plasma retinol, alpha-tocopherol, retinol-binding protein, prealbumin, and zinc were performed on a group of 58 infants weighing less than 2000 g at birth in an intensive-care nursery. Data were classified by the feeding regimen of the preceding week: parenteral, premature formula, or own mother's milk. Mean plasma-retinol values were less than 20 mcg/dl, the lower limit of normal for adults, with the highest values in the formula-fed group. Retinol-binding protein and prealbumin values were lowest in the parenterally-fed group. Alpha-tocopherol concentrations were consistently maintained at levels higher than 500 mcg/dl only in infants fed their own mother's milk. Mean zinc concentrations above 70 mcg/dl, the lower limit of normal for adults, occurred only in parenterally fed infants. Doubling the recommended vitamin supplement in formula-fed infants did not produce a significant increase in plasma retinol or tocopherol.