By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys(358)Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.

PERSPECTIVES were cultured in different glucose concentrations. The amplitude of circadian oscillations in gene expression correlated to glucose concentrations only in wild-type cells, but not in the absence of AMPK. In mouse liver, the accumulation and nuclear localization of AMPK, as well as the phosphorylation of known AMPK target proteins, oscillated in a circadian manner. Thus, perturbation of nutrient availability-and consequently, of AMPK activity-alters output of the circadian clock. Although AMPK is an attractive candidate for coupling metabolic and circadian cycles, additional regulators are likely involved. Thus, the ratio of oxidized nicotinamide ade-nine dinucleotide phosphate (NADP +) to its reduced form (NADPH)-which, like the AMP/ATP ratio, constitutes a diagnostic signature of a cell's metabolic state-has been proposed to affect circadian gene expression through diverse mechanisms. At least in vitro, the binding of the heterodimeric core clock transcription factors CLOCK-BMAL1 and NPAS2-BMAL1 to their cognate DNA sequences (so-called E-boxes) is enhanced by NADPH and impaired by NADP + (6). The transcriptional regulatory protein peroxisome proliferator-activated receptor γ (P PA R γ) coactivator 1α (PGC-1α), a well-known mediator of glucose and lipid metabolism, has been proposed to be another important player in connecting metabolism to circadian gene expression. This transcriptional coacti-vator associates with nuclear receptors of the ROR family and thereby modulates the transcription of the clock genes Bmal1 and Rev-erbα. Finally, the NAD +-dependent protein deacetylase sirtuin 1 infl uences the stability and activity of the core clock components PER2 and BMAL1, respectively (7, 8). Why are metabolic processes under tight circadian control? A simple explanation arises from the necessity to separate incompatible enzymatic processes within the same cell. Because complete spatial separation of anabolic and catabolic processes is frequently impossible, these have to be gated to different time windows. This necessity is well illustrated by the temporal sequestration of oxida-tive and reductive phases in yeast by an ultra-dian respiratory clock. For example, DNA is replicated exclusively in the reductive phase, when the concentration of genotoxic reactive oxygen species generated by mitochondrial respiration is minimal (9). In a yeast mutant in which the reductive phase is too short to allow for the completion of DNA synthesis, the mutation rate increases dramatically (10). In mammals, the master pacemaker in the SCN is phase-entrained primarily by light-dark cycles and thus cannot readily adapt to altered feeding rhythms. Hence, when food availability changes, nutrient-dependent synchronization cues must dominate the more direct signals from the SCN to maintain proper homeostasis of metabolism in peripheral tissues (1). This could explain the multitude of metabolic phase entrainment cues that synchronize the circadian core clock machinery in metabolically active peripheral organs. A major challen...

Sleep is a fundamental biological rhythm involving the interaction of numerous brain structures and diverse neurotransmitter systems. The primary measures used to define sleep are the electroencephalogram (EEG) and electromyogram (EMG). However, EEG-based methods are often unsuitable for use in high-throughput screens as they are both time-intensive and involve invasive surgery. As such, the dissection of sleep mechanisms and the discovery of novel drugs that modulate sleep would benefit greatly from further development of rapid behavioural assays to assess sleep in animal models. Here we describe an automated non-invasive approach to evaluate sleep duration, latency and fragmentation using video tracking of mice in their home cage. This approach provides a high correlation with EEG/EMG measures under both baseline conditions and following administration of pharmacological agents. Moreover, the dose-dependent effects of sedatives, stimulants and light can be readily detected. This approach is both robust yet relatively inexpensive to implement, and can be easily incorporated into ongoing screening programmes to provide a powerful first-pass screen for assessing sleep and allied behaviours.

SummarySleep and circadian rhythm disruption has been widely observed in neuropsychiatric disorders including schizophrenia [1] and often precedes related symptoms [2]. However, mechanistic basis for this association remains unknown. Therefore, we investigated the circadian phenotype of blind-drunk (Bdr), a mouse model of synaptosomal-associated protein (Snap)-25 exocytotic disruption that displays schizophrenic endophenotypes modulated by prenatal factors and reversible by antipsychotic treatment [3, 4]. Notably, SNAP-25 has been implicated in schizophrenia from genetic [5–8], pathological [9–13], and functional studies [14–16]. We show here that the rest and activity rhythms of Bdr mice are phase advanced and fragmented under a light/dark cycle, reminiscent of the disturbed sleep patterns observed in schizophrenia. Retinal inputs appear normal in mutants, and clock gene rhythms within the suprachiasmatic nucleus (SCN) are normally phased both in vitro and in vivo. However, the 24 hr rhythms of arginine vasopressin within the SCN and plasma corticosterone are both markedly phase advanced in Bdr mice. We suggest that the Bdr circadian phenotype arises from a disruption of synaptic connectivity within the SCN that alters critical output signals. Collectively, our data provide a link between disruption of circadian activity cycles and synaptic dysfunction in a model of neuropsychiatric disease.

Background: The properties of vascular endothelial growth factor (VEGF) as a potent vascular permogen and mitogen have led to investigation of its potential role in lung injury. Alternate spliced VEGF transcript generates several isoforms with potentially differing functions. The purpose of this study was to determine VEGF isoform expression and source in normal and ARDS subjects and investigate the expression and regulation of VEGF isoforms by human alveolar type 2 (ATII) cells.

Membrane-associated TNF-alpha cleavage is required to yield the 17.5-kD soluble product. This process is poorly understood in human cells, and no studies have related this process to the alveolar macrophage (AM). TNF-alpha-converting enzyme (TACE) is known to cleave TNF at the Ala-76-Val-77 site. We have evaluated the expression, regulation, and catalytic function of TACE in healthy human AMs. TACE was detected on the surface of AMs using flow cytometry. TACE protein can be upregulated by LPS (P = 0.036) and IFN-gamma. LPS-induced expression is downregulated by IL-10 (P = 0.04) and TNF-alpha. TACE regulation was observed at the mRNA level. TACE catalytic activity as assessed by cleavage of glutathione S-transferase-proTNF fusion protein correlates significantly with TACE protein expression (P = 0.04). However, cleavage and soluble TNF-alpha release by AMs was inhibited by matrix metalloproteinase and serine protease inhibitors, suggesting a role for a serine protease in this process. We confirmed the presence of proteinase-3 (PR-3) on the AM surface that was functionally capable of TNF cleavage. PR-3 mRNA expression was not found in AMs. However, we determined that PR-3 from neutrophil supernatants could bind to the AM membrane, suggesting that AM-derived PR-3 is from an exogenous source, which is important in the context of inflammation.

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