Characterization of a p75NTR Apoptotic Signaling Pathway Using a Novel Cellular Model

The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases

Identification and functional characterization of DR6, a novel death domain‐containing TNF receptor

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116991/1/feb2s0014579398007911.pd

Bimolecular fluorescence complementation analysis of eukaryotic fusion products

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90391/1/BC20100033.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/90391/2/boc_525_sm_suppl.pd

Isolation of genes as chromatin by nucleoprotein hybridization.

The developmentally regulated sea urchin early histone gene repeat (SUEHGR) from Strongylocentrotus purpuratus was isolated as chromatin by nucleoprotein hybridization. This technique is a novel method to isolate specific sequences as chromatin. Because the purification scheme is based on the gene sequence, I was able to isolate the same gene in different functional states. Gene size chromatin fragments were solubilized by restriction endonuclease digestion of cell nuclei. Using T7 gene 6 exonuclease, the 3\sp\prime termini of the fragments were exposed and then hybridized in solution to a biotinylated oligonucleotide complementary to one end of the SUEHGR fragment. The hybrids were bound to an Avidin D matrix and eluted by DTT cleavage of the biotin linker. The inactive genes could be enriched more than 700 fold in SUEHGR sequences in overall yields between 2-15% ($>$80% purity) and could be studied in the electron microscope. The purity of the active gene repeat was \sp\sim15% and yields were 1-3%. The changes occurring during isolation were characterized by micrococcal nuclease digestion, and MPE cleavage combined with indirect end-labeling. The MPE experiments were quantitatively analyzed by the use of an algorithm to calculate cleavage probabilities from the DNA size distribution. The changes in MPE pattern occurring during isolation on the inactive SUEHGR are interpreted as loss of non-histone proteins, while the positions of nucleosomes remain unchanged. Positioned nucleosomes were not found on the active gene repeat, and the only change observed was cutting by site specific and nonspecific endogenous nucleases. Two dimensional gel analysis of the copurified histone and non-histone proteins was made possible by the optimization of an in vitro iodination method. The inactive SUEHGR was depleted in late histone variants as compared to the bulk of the same developmental stage revealing an unequal distribution of histone sequence variants over the genome. Preliminary data indicate a different histone content for the active SUEHGR preparation. The active fraction was enriched in several histone H2A variants, depleted in H3 and H4, and several histones were hyperacetylated. The active and inactive SUEHGR also contained different amounts of a few low molecular weight proteins.Ph.D.BiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103000/1/9227018.pdfDescription of 9227018.pdf : Restricted to UM users only

Changes in the Distributions and Dynamics of Polycomb Repressive Complexes during Embryonic Stem Cell Differentiation▿ †

Polycomb group (PcG) transcription regulatory proteins maintain cell identity by sustained repression of numerous genes. The differentiation of embryonic stem (ES) cells induces a genome-wide shift in PcG target gene expression. We investigated the effects of differentiation and protein interactions on CBX family PcG protein localization and dynamics by using fluorescence imaging. In mouse ES cells, different CBX proteins exhibited distinct distributions and mobilities. Most CBX proteins were enriched in foci known as Polycomb bodies. Focus formation did not affect CBX protein mobilities, and the foci dispersed during ES cell differentiation. The mobilities of CBX proteins increased upon the induction of differentiation and decreased as differentiation progressed. The deletion of the chromobox, which mediates interactions with RING1B, prevented the immobilization of CBX proteins. In contrast, the deletion of the chromodomain, which can bind trimethylated lysine 27 of histone H3, had little effect on CBX protein dynamics. The distributions and mobilities of most CBX proteins corresponded to those of CBX-RING1B complexes detected by using bimolecular fluorescence complementation analysis. Epigenetic reprogramming during ES cell differentiation is therefore associated with global changes in the subnuclear distributions and dynamics of CBX protein complexes

Risk factors for placental malaria, sulfadoxine-pyrimethamine doses, and birth outcomes in a rural to urban prospective cohort study on the Bandiagara Escarpment and Bamako, Mali

Abstract Background Malaria in Mali remains a primary cause of morbidity and mortality, with women at high risk during pregnancy for placental malaria (PM). Risk for PM and its association with birth outcomes was evaluated in a rural to urban longitudinal cohort on the Bandiagara Escarpment and the District of Bamako. Methods Placental samples (N = 317) were collected from 249 mothers who were participants in a prospective cohort study directed by BIS in the years 2011 to 2019. A placental pathologist and research assistant evaluated the samples by histology in blinded fashion to assess PM infection stage and parasite density. Generalized estimating equations (GEE) were used to model the odds of PM infection. Results In a multivariable model, pregnancies in Bamako, beyond secondary education, births in the rainy season (instead of the hot dry season), and births to women who had ≥ 3 doses of sulfadoxine-pyrimethamine (SP) instead of no doses were associated with reduced odds of experiencing PM (active and past infections combined). Births in later years of the study were strongly associated with reduced odds of PM. Maternal age, which was positively associated with offspring year of birth, was significant as a predictor of PM only if offspring year of birth was omitted from the model. Gravidity was positively associated with both maternal age and offspring year of birth such that if either variable was included in the model, then gravidity was no longer significant. However, if maternal age or year of offspring birth were not adjusted for, then the odds of PM were nearly two-fold higher in primigravida compared to multigravida. Birth outcomes improved (+ 285 g birth weight, + 2 cm birth length, + 75 g placental weight) for women who had ≥ 3 doses of SP compared to no doses, but no difference was detected in birth weight or length for women who had 2 instead of ≥ 3 SP doses. However, at 2 instead of ≥ 3 doses placentas were 36 g lighter and the odds of low birth weight ( 10% erythrocytes infected) were significantly associated with decreases in birth weight, birth length, and placental weight, as were chronic PM infections. The women who received no SP during pregnancy (7% of the study total) were younger and lacked primary school education. The women who received ≥ 3 doses of SP came from more affluent families. Conclusions Women who received no doses of SP during pregnancy experienced the most disadvantageous birth outcomes in both Bamako and on the Bandiagara Escarpment. Such women tended to be younger and to have had no primary school education. Targeting such women for antenatal care, which is the setting in which SP is most commonly administered in Mali, will have a more positive impact on public health than focusing on the increment from two to three doses of SP, although that increment is also desirable.http://deepblue.lib.umich.edu/bitstream/2027.42/173706/1/12936_2022_Article_4125.pd