Faculty Bibliography

The N-ribosyldihydronicotinamide (NRH):quinone oxidoreductase 2 (NQO2) gene encodes an enzyme that catalyzes activation of quinones. Blood DNA from 80 control individuals and 118 age-matched Parkinson's disease patients were analyzed for NQO2 gene promoter polymorphisms. The results revealed three allelic variants, designated I-29, I-16, and D. These results were confirmed in fibroblast cell lines. In patients with Parkinson's disease, there was a significant increase in the frequency of the D allele, but there was no difference in the frequency of the alleles in familial compared to sporadic Parkinson's disease. The D and I-16 promoters direct higher NQO2 gene expression that results in higher enzyme activity. Overexpression of NQO2 in the catecholaminergic neuroblastoma SH-SY5Y cells resulted in increased production of reactive oxygen species when exposed to exogenous dopamine. The results suggest that the association of the D promoter with Parkinson's disease may be due to an increase in expression of the NQO2 gene.

In this study, the complete genomic sequence was determined for three hepatitis C virus variants (VT21, TV241 and TV249) of genotype 6 that do not classify within the established subtypes. All three genomes were isolated from patients in Vietnam and sequenced using 100 microl of serum. They showed 91.4-93.6% nucleotide similarities to each other but only 71.7-79.4% similarities to 17 reference sequences representing subtypes 6a-6q and to isolates km41 and gz52557. VT21, TV241 and TV249 displayed genome lengths of 9407, 9460 and 9445 nt, respectively. All three isolates contained a single open reading frame of 9051 nt while the 5'UTRs and 3'UTRs were 324-338 nt and 32-71 nt, respectively. They shared common sizes with QC227/6o and QC216/6p isolates in all ten protein regions. Phylogenetic analyses demonstrated that VT21, TV241 and TV249 clustered independently and were assigned subtype 6t, following the recent designations of 6r and 6s. Analysis of partial genomic sequences available for genotype 6 variants revealed five additional subtype 6t isolates, all originating from Vietnam. This analysis revealed two additional groups of isolates, and at least seven novel variants analogous to km41 and gz52557 that group independently and do not classify within the subtypes 6a-6t. This suggests the existence of at least 11 additional subtypes for genotype 6. In addition, the existence of isolates showing genetic distances greater than those within subtypes, but lesser than those between subtypes, raises interesting questions regarding the classification of HCV.

Responses to psychostimulants vary with age, but the molecular etiologies of these differences are largely unknown. The goal of the present research was to identify age-specific behavioral and molecular adaptations to cocaine and to elucidate the mechanisms involved therein. Postweanling, periadolescent, and adult male CD-1 mice were exposed to cocaine (20 mg/kg) for 7 d. The rewarding effects of cocaine were assessed, as were the response to a Trk antagonist and the regulation of dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32). Cocaine was rewarding in both periadolescent and adult mice using a conditioned place preference procedure. In contrast, postweanling mice failed to demonstrate significant cocaine-induced place preference. Because components of the neurotrophin system including brain-derived neurotrophic factor and TrkB are developmentally regulated, their role in the age-specific effects of cocaine was determined using the Trk receptor antagonist K252a. Postweanling mice that received K252a before daily cocaine showed a significant place preference to the cocaine-paired environment that was not seen in the absence of K252a. DARPP-32 protein levels were significantly upregulated in the lateral region of the caudate-putamen exclusively in postweanling mice after chronic cocaine. Daily pretreatment with K252a attenuated the induction of DARPP-32 in the postweanling striatum. These data indicate that Trk neurotransmission plays a role in age-specific behavioral and molecular responses to cocaine and concurrently modulates DARPP-32 levels.

Innate immunity is the first-line defense against pathogens and relies on phagocytes, soluble components, and cell-surface and cytosolic pattern recognition receptors. Despite using hard-wired receptors and signaling pathways, the innate immune response demonstrates surprising specificity to different pathogens. We determined how combinatorial use of innate immune defense mechanisms defines the response. We describe a novel cooperation between a soluble component of the innate immune system, the mannose-binding lectin, and Toll-like receptor 2 that both specifies and amplifies the host response to Staphylococcus aureus. Furthermore, we demonstrate that this cooperation occurs within the phagosome, emphasizing the importance of engulfment in providing the appropriate cellular environment to facilitate the synergy between these defense pathways

Prohibitin 1 (PHB1) is a highly conserved protein that is mainly localized to the inner mitochondrial membrane and has been implicated in regulating mitochondrial function in yeast. Because mitochondria are emerging as an important regulator of vascular homeostasis, we examined PHB1 function in endothelial cells. PHB1 is highly expressed in the vascular system and knockdown of PHB1 in endothelial cells increases mitochondrial production of reactive oxygen species via inhibition of complex I, which results in cellular senescence. As a direct consequence, both Akt and Rac1 are hyperactivated, leading to cytoskeletal rearrangements and decreased endothelial cell motility, e.g., migration and tube formation. This is also reflected in an in vivo angiogenesis assay, where silencing of PHB1 blocks the formation of functional blood vessels. Collectively, our results provide evidence that PHB1 is important for mitochondrial function and prevents reactive oxygen species-induced senescence and thereby maintains the angiogenic capacity of endothelial cells

Drosophila neuroblasts are similar to mammalian neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. In the past two decades, great advances have been made in understanding the molecular mechanisms underlying embryonic neuroblast formation, the establishment of cell polarity and the temporal regulation of cell fate. It is now a challenge to connect, at the molecular level, the different cell biological events underlying the transition from neural stem cell maintenance to differentiation. Progress has also been made in understanding the later stages of development, when neuroblasts become mitotically inactive, or quiescent, and are then reactivated postembryonically to generate the neurons that make up the adult nervous system. The ability to manipulate the steps leading from quiescence to proliferation and from proliferation to differentiation will have a major impact on the treatment of neurological injury and neurodegenerative disease.

The long-awaited structure of the effector portion of IRE1, the endoplasmic reticulum stress transducer, is published in this issue of Cell (Lee et al., 2008). This structure provides new insight into the mysterious coupling of kinase and endoribonuclease activities in the oldest, most-conserved branch of the unfolded protein response in eukaryotes

Notch signaling controls diverse eukaryotic differentiation processes in multiple cell types, thus demanding versatile tools with which Notch triggers downstream events. Ubiquitin-mediated proteolysis has previously been shown to be one such tool with which Notch regulates the turnover of the basic helix-loophelix transcription factor, E47. Here, we show that Notch signaling also accelerated the degradation of Tal1/SCL (T cell acute leukemia 1/stem cell leukemia) protein, a basic helix-loop-helix protein involved in the development of hematopoietic, vascular, and neuronal tissues. Notch-induced Tal1/SCL degradation was mediated by ubiquitination and proteasomes. The sequence responsible for Tal1 degradation was localized to a region in the C terminus of Tal1, which is evolutionarily conserved, thus suggesting a functional significance. Analogous to the situation for E47, Notch-induced Tal1/SCL degradation not only required Skp2, a substrate-binding subunit of SCF ubiquitin ligase complexes, but also relied on CHIP, a chaperone-binding protein with a ubiquitin ligase activity. In contrast to the fact that the N-terminal tetratricopeptide region (TPR) domain of CHIP is necessary and sufficient for E47 ubiquitination and degradation, CHIP promoted Tal1 degradation with both chaperone binding and ubiquitin ligase activities, which are mediated by its TPR domain and U box, respectively. Although the TPR domain was not involved in Tal1/SCL binding, it was required for enhancing its degradation. Likewise, the ubiquitin ligase activity of CHIP was dispensable for Tal1/SCL binding but essential for degradation. These findings provide both novel mechanistic insights into the operation of cullin-based ubiquitin ligase complexes and potential means by which Notch and Tal1/SCL regulate eukaryotic development.

MicroRNAs (miRNAs) are highly expressed in vertebrate neural tissues, but the contribution of specific miRNAs to the development and function of different neuronal populations is still largely unknown. We report that miRNAs are required for terminal differentiation of olfactory precursors in both mouse and zebrafish but are dispensable for proper function of mature olfactory neurons. The repertoire of miRNAs expressed in olfactory tissues contains over 100 distinct miRNAs. A subset, including the miR-200 family, shows high olfactory enrichment and expression patterns consistent with a role during olfactory neurogenesis. Loss of function of the miR-200 family phenocopies the terminal differentiation defect observed in absence of all miRNA activity in olfactory progenitors. Our data support the notion that vertebrate tissue differentiation is controlled by conserved subsets of organ-specific miRNAs in both mouse and zebrafish and provide insights into control mechanisms underlying olfactory differentiation in vertebrates.

The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2alpha at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2alpha. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2alpha in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress