The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction.

The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.

Expression and regulation of mPer1 in immortalized GnRH neurons.

Hypothalamic GnRH (gonadotropin-releasing hormone) neurons play a critical role in the initiation and maintenance of reproduction competence. Using the mouse GnRH neuronal cell line, GT1-7, we have characterized the expression of the gene mPer1, a recognized key element of the mammalian circadian clockwork. Both mPer1 transcripts and the 136 kDa mPER1 gene product could be detected in these cells. Immunocytochemical analysis also confirmed expression of mPER1 both in vitro and in vivo in GnRH neurons. Activation of cyclic AMP signalling pathways in vitro elevated GnRH secretion as well as mPer1 expression and nuclear mPER1 immunoreactivity. As mPER1 is known to feedback on transcriptional activities in many cell models, the data presented here point to a role for mPER1 in the regulation of gene expression in GnRH neurons, and thus in the control of neuroendocrine activities.

Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock.

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice were immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.

Postmortem stability of melatonin receptor binding and clock-relevant mRNAs in mouse suprachiasmatic nucleus.

The stability of receptor proteins and mRNAs in brain tissue is variable after death. As a prelude to quantitative studies of melatonin receptor density and clock gene expression in the human brain, the stability of these macromolecules was examined in the mouse brain under simulated postmortem conditions using the model of Spokes and Koch. In the mouse suprachiasmatic nucleus (SCN), melatonin receptor binding was significantly reduced after 18 to 24 h under postmortem conditions. Two mRNAs that are rhythmically expressed in the SCN, mPer1 and prepropressophysin (AVP), also decreased significantly over the interval studied, and mPer1 declined more rapidly than AVP. Both mPer1 and AVP mRNA levels in the SCN declined more rapidly in vivo than under postmortem conditions, suggesting that the degradation of these mRNAs is an active process. The results indicate that quantitative studies of melatonin receptor density on human postmortem material are feasible and that detection of rhythmic gene expression in the human SCN will likely require collection of specimens with a rather short (< 8 h) interval from death to tissue collection. The relative stability of melatonin receptor binding in the SCN also suggests that receptor binding may be a reliable marker for the location of the SCN in studies assessing clock gene expression in postmortem material.

Molecular analysis of mammalian circadian rhythms.

In mammals, a master circadian "clock" resides in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN clock is composed of multiple, single-cell circadian oscillators, which, when synchronized, generate coordinated circadian outputs that regulate overt rhythms. Eight clock genes have been cloned that are involved in interacting transcriptional-/translational-feedback loops that compose the molecular clockwork. The daily light-dark cycle ultimately impinges on the control of two clock genes that reset the core clock mechanism in the SCN. Clock-controlled genes are also generated by the central clock mechanism, but their protein products transduce downstream effects. Peripheral oscillators are controlled by the SCN and provide local control of overt rhythm expression. Greater understanding of the cellular and molecular mechanisms of the SCN clockwork provides opportunities for pharmacological manipulation of circadian timing.

Distinct pharmacological mechanisms leading to c-fos gene expression in the fetal suprachiasmatic nucleus.

Maternal treatment with cocaine or a D1-dopamine receptor agonist induces c-fos gene expression in the fetal suprachiasmatic nuclei (SCN). Other treatments that induce c-fos expression in the fetal SCN include caffeine and nicotine. In the current article, the authors assessed whether these different pharmacological treatments activate c-fos expression by a common neurochemical mechanism. The results indicate the presence of at least two distinct pharmacological pathways to c-fos expression in the fetal rat SCN. Previous studies demonstrate that prenatal activation of dopamine receptors affects the developing circadian system. The present work shows that stimulant drugs influence the fetal brain through multiple transmitter systems and further suggests that there may be multiple pathways leading to entrainment of the fetal biological clock.

Targeted disruption of the mPer3 gene: subtle effects on circadian clock function.

Neurons in the mammalian suprachiasmatic nucleus (SCN) contain a cell-autonomous circadian clock that is based on a transcriptional-translational feedback loop. The basic helix-loop-helix-PAS proteins CLOCK and BMAL1 are positive regulators and drive the expression of the negative regulators CRY1 and CRY2, as well as PER1, PER2, and PER3. To assess the role of mouse PER3 (mPER3) in the circadian timing system, we generated mice with a targeted disruption of the mPer3 gene. Western blot analysis confirmed the absence of mPER3-immunoreactive proteins in mice homozygous for the targeted allele. mPer1, mPer2, mCry1, and Bmal1 RNA rhythms in the SCN did not differ between mPER3-deficient and wild-type mice. Rhythmic expression of mPer1 and mPer2 RNAs in skeletal muscle also did not differ between mPER3-deficient and wild-type mice. mPer3 transcripts were rhythmically expressed in the SCN and skeletal muscle of mice homozygous for the targeted allele, but the level of expression of the mutant transcript was lower than that in wild-type controls. Locomotor activity rhythms in mPER3-deficient mice were grossly normal, but the circadian cycle length was significantly (0.5 h) shorter than that in controls. The results demonstrate that mPer3 is not necessary for circadian rhythms in mice.

Melatonin limits transcriptional impact of phosphoCREB in the mouse SCN via the Mel1a receptor.

In the mouse, activity phase-shifts of the endogenous clock in the suprachiasmatic nucleus (SCN) are associated with phosphorylation of the transcription factor Ca2+/cAMP responsive element binding protein (CREB). CREB phosphorylation is induced by the retino-hypothalamic transmitter pituitary adenylate cyclase-activating polypeptide (PACAP). As detected by immunohistochemistry in SCN slices from wild-type mice, melatonin completely blocked PACAP-stimulated CREB phosphorylation at low concentrations (1 nM). In Mel1a melatonin receptor-deficient mice, the PACAP-induced CREB phosphorylation was inhibited only at melatonin concentrations of 100 nM. This inhibition was, however, blunted by blocking the Mel1b melatonin receptor. Thus, melatonin modulates PACAP-mediated retinal stimuli for clock entrainment primarily via the Mel1a melatonin receptor through molecular interaction within the cAMP-signalling pathway.

A time-less function for mouse timeless.

The timeless (tim) gene is essential for circadian clock function in Drosophila melanogaster. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Here we show that mTim is essential for embryonic development, but does not have substantiated circadian function.

Interacting molecular loops in the mammalian circadian clock.

We show that, in the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2(Brdm1) mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with a dominant role of PERIOD2 in the positive regulation of the Bmal1 loop. In vitro analysis of CRYPTOCHROME inhibition of CLOCK: BMAL1-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the PERIOD and TIMELESS proteins. PERIOD2 is a positive regulator of the Bmal1 loop, and CRYPTOCHROMES are the negative regulators of the Period and Cryptochrome cycles.

Analysis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the circadian clockwork and resetting mechanisms.

The circadian clock in the suprachiasmatic nuclei (SCN) is comprised of a cell-autonomous, autoregulatory transcriptional/translational feedback loop. Its molecular components include three period and two cryptochrome genes. We describe circadian patterns of expression of mPER2 and mPER3 in the mouse SCN that are synchronous to those for mPER1, mCRY1, and mCRY2. Coimmunoprecipitation experiments demonstrate in vivo associations of the SCN mPER proteins with each other and with the mCRY proteins, and of mCRY proteins with mTIM, but no mPER/mTIM interactions. Examination of the effects of weak and strong resetting light pulses on SCN clock proteins highlights a central role for mPER1 in photic entrainment, with no acute light effects on either the mCRY or mTIM proteins. These clock protein interactions and photic responses in mice are divergent from those described in Drosophila.

mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.

We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

Differential regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new insights into a core clock mechanism.

Recent discoveries have identified a framework for the core circadian clock mechanism in mammals. Development of this framework has been based entirely on the expression patterns of so-called "clock genes" in the suprachiasmatic nuclei (SCN), the principal clock of mammals. We now provide data concerning the protein expression patterns of two of these genes, mPer1 and mTim. Our studies show that mPER1 and mTIM are nuclear antigens expressed in the SCN and extensively throughout the forebrain. Expression of mPER1 in the SCN was rhythmic under entrained conditions and with clear circadian cycling under free-running conditions. Expression of mPER1 elsewhere in the mouse forebrain was not rhythmic. In contrast to mPER1, mTIM expression in the SCN did not vary with time in mice housed in either a light/dark cycle or in constant dim red light. The phase relationship between mPer1 RNA and mPER1 cycles in the SCN is consistent with a negative feedback model of the mammalian clock. The invariant nature of nuclear mTIM in the SCN suggests that its participation in negative feedback occurs only after mPER1 has entered the nucleus, and that the abundance of mTIM is not regulated by the circadian clock or the light/dark cycle.

Photic induction of Period gene expression is reduced in Clock mutant mice.

The Clock mutation leads to abnormal circadian behavior and defective transcriptional activity of CLOCK, a basic helix-loop-helix (bHLH)/PAS protein. In situ hybridization was used to assess whether the Clock mutation affects the photic induction of mPer1, mPer2, and c-fos in the mouse suprachiasmatic nucleus (SCN). Exposure of wild-type mice to a 15 min light pulse at night rapidly induced expression of c-fos mRNA, with mPer1 and mPer2 mRNAs peaking later. Light exposure also increased c-fos, mPer1 and mPer2 mRNA levels in the SCN of homozygous Clock mutant mice, but the amplitude of the response to light was significantly reduced. Clock appears to play a role in circadian photoreception that is distinct from its role in the circadian oscillatory mechanism.

Expression of basic helix-loop-helix/PAS genes in the mouse suprachiasmatic nucleus.

The suprachiasmatic nuclei contain a circadian clock that drives rhythmicity in physiology and behavior. In mice, mutation of the Clock gene produces abnormal circadian behavior [Vitaterna M. H. et al. (1994) Science 264, 715-725]. The Clock gene encodes a protein containing basic helix-loop-helix and PAS (PER-ARNT-SIM) domains [King D. P. et al. (1997) Cell 89, 641-653]. The PAS domain may be an important structural feature of a subset of genes involved in photoreception and circadian rhythmicity. The expression and regulation of messenger RNAs encoding eight members of the basic helix-loop-helix/PAS protein superfamily were examined by in situ hybridization. Six of the genes studied (aryl hydrocarbon receptor nuclear transporter, aryl hydrocarbon receptor nuclear transporter-2, Clock, endothelial PAS-containing protein, hypoxia-inducible factor-1alpha and steroid receptor coactivator-1) were expressed in the suprachiasmatic nucleus of adult and neonatal mice. No evidence for rhythmicity of expression was observed when comparing brains collected early in the subjective day (circadian time 3) with those collected early in subjective night (circadian time 15). Neuronal PAS-containing protein-1 messenger RNA was expressed in the suprachiasmatic nucleus of adult (but not neonatal) mice, and a low-amplitude rhythm of neuronal PAS-containing protein-1 gene expression was detected in the suprachiasmatic nucleus. Neuronal PAS-containing protein-2 messenger RNA was not detected in adult or neonatal suprachiasmatic nucleus. Exposure to light at night (30 or 180 min of light, beginning at circadian time 15) did not alter the expression of any of the genes studied. The expression of multiple members of the basic helix-loop-helix/PAS family in the suprachiasmatic nucleus suggests a rich array of potential interactions relevant to the regulation of the suprachiasmatic circadian clock.

A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock.

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

Molecular analysis of mammalian timeless.

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.

In-phantom neutron fluence measurements in the orthogonal Birmingham boron neutron capture therapy beam.

This paper presents the results of an experimental investigation into the performance of the Birmingham accelerator-based epithermal BNCT beam. In-phantom gold foil activation and boron trifluoride tube measurements have been used. The results have been compared with calculated response rates using Monte Carlo modeling of the entire neutron system from source to phantom and detector. The excellent agreement obtained gives us confidence in the validity of the simulations and our ability to predict accurately the neutronic performance of our BNCT facility.