Faculty Bibliography

Causes of late Quaternary extinctions of large mammals ('megafauna') continue to be debated, especially for continental losses, because spatial and temporal patterns of extinction are poorly known. Accurate latest appearance dates (LADs) for such taxa are critical for interpreting the process of extinction. The extinction of woolly mammoth and horse in northwestern North America is currently placed at 15,000-13,000 calendar years before present (yr BP), based on LADs from dating surveys of macrofossils (bones and teeth). Advantages of using macrofossils to estimate when a species became extinct are offset, however, by the improbability of finding and dating the remains of the last-surviving members of populations that were restricted in numbers or confined to refugia. Here we report an alternative approach to detect 'ghost ranges' of dwindling populations, based on recovery of ancient DNA from perennially frozen and securely dated sediments (sedaDNA). In such contexts, sedaDNA can reveal the molecular presence of species that appear absent in the macrofossil record. We show that woolly mammoth and horse persisted in interior Alaska until at least 10,500 yr BP, several thousands of years later than indicated from macrofossil surveys. These results contradict claims that Holocene survival of mammoths in Beringia was restricted to ecologically isolated high-latitude islands. More importantly, our finding that mammoth and horse overlapped with humans for several millennia in the region where people initially entered the Americas challenges theories that megafaunal extinction occurred within centuries of human arrival or were due to an extraterrestrial impact in the late Pleistocene

Intracellular lipid accumulation in the heart is associated with cardiomyopathy, yet the precise role of triglyceride (TG) remains unclear. With exercise, wild type hearts develop physiologic hypertrophy. This was associated with greater TG stores and a marked induction of the TG-synthesizing enzyme diacylglycerol (DAG) acyltransferase 1 (DGAT1). Transgenic overexpression of DGAT1 in the heart using the cardiomyocyte- specific alpha-myosin heavy chain (MHC) promoter led to approximately a doubling of DGAT activity and TG content and reductions of approximately 35% in cardiac ceramide, 26% in DAG, and 20% in free fatty acid levels. Cardiac function assessed by echocardiography and cardiac catheterization was unaffected. These mice were then crossed with animals expressing long-chain acyl-CoA synthetase via the MHC promoter (MHC-ACS), which develop lipotoxic cardiomyopathy. MHC-DGAT1XMHC-ACS double transgenic male mice had improved heart function; fractional shortening increased by 74%, and diastolic function improved compared with MHC-ACS mice. The improvement of heart function correlated with a reduction in cardiac DAG and ceramide and reduced cardiomyocyte apoptosis but increased fatty acid oxidation. In addition, the survival of the mice was improved. Our study indicates that TG is not likely to be a toxic lipid species directly, but rather it is a feature of physiologic hypertrophy and may serve a cytoprotective role in lipid overload states. Moreover, induction of DGAT1 could be beneficial in the setting of excess heart accumulation of toxic lipids.

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc(alpha), Alc(beta), and Alc(gamma). The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP alpha-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent gamma-secretase complex, thereby generating 'APP p3-like' and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma), whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor beta-amyloid species Abeta42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma) were not equivalent, suggesting that one type of gamma-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect gamma-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects

BACKGROUND: Recent reports suggest that latrepirdine (Dimebon, dimebolin), a retired Russian antihistamine, improves cognitive function in aged rodents and in patients with mild to moderate Alzheimer's disease (AD). However, the mechanism(s) underlying this benefit remain elusive. AD is characterized by extracellular accumulation of the amyloid-beta (Abeta) peptide in the brain, and Abeta-lowering drugs are currently among the most popular anti-amyloid agents under development for the treatment of AD. In the current study, we assessed the effect of acute dosing of latrepirdine on levels of extracellular Abeta using in vitro and in vivo experimental systems. RESULTS: We evaluated extracellular levels of Abeta in three experimental systems, under basal conditions and after treatment with latrepirdine. Mouse N2a neuroblastoma cells overexpressing Swedish APP were incubated for 6 hr in the presence of either vehicle or vehicle + latrepirdine (500pM-5 muM). Synaptoneurosomes were isolated from TgCRND8 mutant APP-overexpressing transgenic mice and incubated for 0 to 10 min in the absence or presence of latrepirdine (1 muM or 10 muM). Drug-naive Tg2576 Swedish mutant APP overexpressing transgenic mice received a single intraperitoneal injection of either vehicle or vehicle + latrepirdine (3.5 mg/kg). Picomolar to nanomolar concentrations of acutely administered latrepirdine increased the extracellular concentration of Abeta in the conditioned media from Swedish mutant APP-overexpressing N2a cells by up to 64% (p = 0.01), while a clinically relevant acute dose of latrepirdine administered i.p. led to an increase in the interstitial fluid of freely moving APP transgenic mice by up to 40% (p = 0.01). Reconstitution of membrane protein trafficking and processing is frequently inefficient, and, consistent with this interpretation, latrepirdine treatment of isolated TgCRND8 synaptoneurosomes involved higher concentrations of drug (1-10 muM) and led to more modest increases in extracellular Abeta(x-42 )levels (+10%; p = 0.001); of note, however, was the observation that extracellular Abeta(x-40 )levels did not change. CONCLUSIONS: Here, we report the surprising association of acute latrepirdine dosing with elevated levels of extracellular Abeta as measured in three independent neuron-related or neuron-derived systems, including the hippocampus of freely moving Tg2576 mice. Given the reported association of chronic latrepirdine treatment with improvement in cognitive function, the effects of chronic latrepirdine treatment on extracellular Abeta levels must now be determined

Neural stem cells must strike a balance between self-renewal and multipotency, and differentiation. Identification of the transcriptional networks regulating stem cell division is an essential step in understanding how this balance is achieved. We have shown that the homeodomain transcription factor, Prospero, acts to repress self-renewal and promote differentiation. Among its targets are three neural stem cell transcription factors, Asense, Deadpan and Snail, of which Asense and Deadpan are repressed by Prospero. Here, we identify the targets of these three factors throughout the genome. We find a large overlap in their target genes, and indeed with the targets of Prospero, with 245 genomic loci bound by all factors. Many of the genes have been implicated in vertebrate stem cell self-renewal, suggesting that this core set of genes is crucial in the switch between self-renewal and differentiation. We also show that multiply bound loci are enriched for genes previously linked to nervous system phenotypes, thereby providing a shortcut to identifying genes important for nervous system development.

Despite a large steric bulk of C(60), a molecular graphene with a covalently linked C(60) pendant [hexabenzocoronene (HBC)-C(60); 1] self-assembles into a coaxial nanotube whose wall consists of a graphite-like pi-stacked HBC array, whereas the nanotube surface is fully covered by a molecular layer of clustering C(60). Because of this explicit coaxial configuration, the nanotube exhibits an ambipolar character in the field-effect transistor output [hole mobility (micro(h)) = 9.7 x 10(-7) cm(2) V(-1) s(-1); electron mobility (micro(e)) = 1.1 x 10(-5) cm(2) V(-1) s(-1)] and displays a photovoltaic response upon light illumination. Successful coassembly of 1 and an HBC derivative without C(60) (2) allows for tailoring the p/n heterojunction in the nanotube, so that its ambipolar carrier transport property can be optimized for enhancing the open-circuit voltage in the photovoltaic output. As evaluated by an electrodeless method called flash-photolysis time-resolved microwave conductivity technique, the intratubular hole mobility (2.0 cm(2) V(-1) s(-1)) of a coassembled nanotube containing 10 mol % of HBC-C(60) (1) is as large as the intersheet mobility in graphite. The homotropic nanotube of 2 blended with a soluble C(60) derivative [(6,6)-phenyl C(61) butyric acid methyl ester] displayed a photovoltaic response with a much different composition dependency, where the largest open-circuit voltage attained was obviously lower than that realized by the coassembly of 1 and 2.

Antioxidants cause dissociation of nuclear factor erythroid 2-related factor 2 (Nrf2) from inhibitor of Nrf2 (INrf2) and so Nrf2:INrf2 can serve as a sensor of oxidative stress. Nrf2 translocates to the nucleus, binds to antioxidant response element (ARE) and activates defensive gene expression, which protects cells. Controversies exist regarding the role of antioxidant-induced modification of INrf2 cysteine 151 or protein kinase C (PKC)-mediated phosphorylation of Nrf2 serine 40 in the release of Nrf2 from INrf2. In addition, the PKC isoform that phosphorylates Nrf2S40 remains unknown. Here, we demonstrate that antioxidant-induced PKC-delta-mediated phosphorylation of Nrf2S40 leads to release of Nrf2 from INrf2. This was evident from specific chemical inhibitors of PKC isoenzymes in reporter assays, in vitro kinase assays with purified Nrf2 and PKC isoenzymes, in vivo analysis with dominant-negative mutants and siRNA against PKC isoforms, use of PKC-delta(+/+) and PKC-delta(-/-) cells, and use of Nrf2S40 phospho-specific antibody. The studies also showed that antioxidant-induced INrf2C151 modification was insufficient for the dissociation of Nrf2 from INrf2. PKC-delta-mediated Nrf2S40 phosphorylation was also required. Nrf2 and mutant Nrf2S40A both bind to INrf2. However, antioxidant treatment led to release of Nrf2 but not Nrf2S40A from INrf2. In addition, Nrf2 and mutant Nrf2S40A both failed to dissociate from mutant INrf2C151A. Furthermore, antioxidant-induced ubiquitylation of INrf2 in PKC-delta(+/+) and PKC-delta(-/-) cells occurred, but Nrf2 failed to be released in PKC-delta(-/-) cells. The antioxidant activation of Nrf2 reduced etoposide-mediated DNA fragmentation and promoted cell survival in PKC-delta(+/+) but not in PKC-delta(-/-) cells. These data together demonstrate that both modification of INrf2C151 and PKC-delta-mediated phosphorylation of Nrf2S40 play crucial roles in Nrf2 release from INrf2, antioxidant induction of defensive gene expression, promoting cell survival, and increasing drug resistance.

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies.

Defects in pRb tumor suppressor pathway occur in approximately 50% of the deadly muscle-invasive urothelial carcinomas in humans and urothelial carcinoma is the most prevalent epithelial cancer in long-term survivors of hereditary retinoblastomas caused by loss-of-function RB1 mutations. Here, we show that conditional inactivation of both RB1 alleles in mouse urothelium failed to accelerate urothelial proliferation. Instead, it profoundly activated the p53 pathway, leading to extensive apoptosis, and selectively induced pRb family member p107. Thus, pRb loss triggered multiple fail-safe mechanisms whereby urothelial cells evade tumorigenesis. Additional loss of p53 in pRb-deficient urothelial cells removed these p53-dependent tumor barriers, resulting in late-onset hyperplasia, umbrella cell nuclear atypia, and rare-occurring low-grade, superficial papillary bladder tumors, without eliciting invasive carcinomas. Importantly, mice deficient in both pRb and p53, but not those deficient in either protein alone, were highly susceptible to subthreshold carcinogen exposure and developed invasive urothelial carcinomas that strongly resembled the human counterparts. The invasive lesions had a marked reduction of p107 but not p130 of the pRb family. Our data provide compelling evidence, indicating that urothelium, one of the slowest cycling epithelia, is remarkably resistant to transformation by pRb or p53 deficiency; that concurrent loss of these two tumor suppressors is necessary but insufficient to initiate urothelial tumorigenesis along the invasive pathway; that p107 may play a critical role in suppressing invasive urothelial tumor formation; and that replacing/restoring the function of pRb, p107, or p53 could be explored as a potential therapeutic strategy to block urothelial tumor progression