Faculty Bibliography

We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P450 (CYP)1A expression for up to 45 days by mechanisms other than persistence of the parent MC (Moorthy, J. 2000. Pharmacology. Exp. Ther. 294, 313-322). The CYP1A genes are members of the Ah gene battery that also encode CYP1B1 and phase II enzymes such as glutathione S-transferase (GST-alpha), UDP glucuronyl transferase (UGT)1A, NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced):quinone oxidoreductase I (NQO1), aldehyde dehydrogenase (ALDH), etc. Therefore, in this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1B1 and phase II genes, which are in part regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 mumol/kg), ip, once daily for 4 days, and expression of CYP1B1 and several phase II (e.g., GST-alpha, NQO1) genes and their corresponding proteins were determined in lung and liver. The major finding was that MC persistently induced (3- to 10-fold) the expression of several phase II enzymes, including GST-alpha, NQO1, UGT1A1, ALDH, and epoxide hydrolase in both tissues for up to 28 days. However, MC did not elicit sustained induction of CYP1B1. Our results thus support the hypothesis that MC elicits coordinated and sustained induction of phase II genes presumably via persistent activation of the AHR, a phenomenon that may have implications for chemical-induced carcinogenesis and chemopreventive strategies in humans.

Glucocorticoids (GCs) display both protective and destructive effects in the nervous system. In excess, GCs produce neuronal damage after stress or brain injury; however, the neuroprotective effects of adrenal steroids also have been reported. The mechanisms that account for the positive actions are not well understood. Here we report that GCs can selectively activate Trk receptor tyrosine kinases after in vivo administration in the brain and in cultures of hippocampal and cortical neurons. Trk receptors are normally activated by neurotrophins, such as NGF and brain-derived neurotrophic factor, but the activation of Trk receptors by GCs does not depend on increased production of neurotrophins. Other tyrosine kinase receptors, such as EGF and FGF receptors, were not activated by GCs. The ability of GCs to increase Trk receptor activity resulted in the neuroprotection of neurons deprived of trophic support and could be modulated by steroid-converting enzymes. Pharmacological and shRNA experiments indicate that Trk receptor activation by GCs depends on a genomic action of the GC receptor. The ability of GCs to promote Trk receptor activity represents a molecular mechanism that integrates the actions of GCs and neurotrophins

Ectopic expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors, we have shown that exogenous factors are required for about 10 days, after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition period. While downregulation of Thy1 and subsequent upregulation of SSEA-1 occur at early time points, reactivation of endogenous Oct4, Sox2, telomerase, and the silent X chromosome mark late events in the reprogramming process. Cell sorting with these markers allows for a significant enrichment of cells with the potential to become iPS cells. Our results suggest that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain the majority of cells poised to become iPS cells

Neurogenesis, the division, migration, and differentiation of new neurons, occurs throughout life. Brain derived neurotrophic factor (BDNF) has been identified as a potential signaling molecule regulating neurogenesis in the subventricular zone (SVZ), but its functional consequences in vivo have not been well defined. We report marked and unexpected deficits in survival but not proliferation of newly born cells of adult knock-in mice containing a variant form of BDNF [a valine (Val) to methionine (Met) substitution at position 66 in the prodomain of BDNF (Val66Met)], a genetic mutation shown to lead to a selective impairment in activity-dependent BDNF secretion. Utilizing knock-out mouse lines, we identified BDNF and tyrosine receptor kinase B (TrkB) as the critical molecules for the observed impairments in neurogenesis, with p75 knock-out mice showing no effect on cell proliferation or survival. We then localized the activated form of TrkB to a discrete population of cells, type A migrating neuroblasts, and demonstrate a decrease in TrkB phosphorylation in the SVZ of Val66Met mutant mice. With these findings, we identify TrkB signaling, potentially through activity dependent release of BDNF, as a critical step in the survival of migrating neuroblasts. Utilizing a behavioral task shown to be sensitive to disruptions in olfactory bulb neurogenesis, we identified specific impairments in spontaneous olfactory discrimination, but not general olfactory sensitivity or habituation to olfactory stimuli in BDNF mutant mice. Through these observations, we have identified novel links between genetic variant BDNF and adult neurogenesis in vivo, which may contribute to significant impairments in olfactory function

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed 'the donut' and appears to inhibit BHT-induced viral envelope fusion

Proteomic analyses of the nucleolus have revealed almost 700 functionally diverse proteins implicated in ribosome biogenesis, nucleolar assembly, and regulation of vital cellular processes. However, this nucleolar inventory has not unveiled a specific consensus motif necessary for nucleolar binding. The ribosomal protein family characterized by their basic nature should exhibit distinct binding sequences that enable interactions with the rRNA precursor molecules facilitating subunit assembly. We succeeded in delineating 2 minimal nucleolar binding sequences of human ribosomal protein S6 by fusing S6 cDNA fragments to the 5' end of the LacZ gene and subsequently detecting the intracellular localization of the beta-galactosidase fusion proteins. Nobis1 (nucleolar binding sequence 1), comprising of 4 highly conserved amino acid clusters separated by glycine or proline, functions independently of the 3 authentic nuclear localization signals (NLSs). Nobis2 consists of 2 conserved peptide clusters and requires the authentic NLS2 in its native context. Similarly, we deduced from previous publications that the single Nobis of ribosomal protein S25 is also highly conserved. The functional protein domain organization of the ribosomal protein S6e family consists of 3 modules: NLS, Nobis, and the C-terminal serine cluster of the phosphorylation sites. This modular structure is evolutionary conserved in vertebrates, invertebrates, and fungi. Remarkably, nucleolar binding sequences of small and large ribosomal proteins reside in peptide clusters conserved over millions of years.