Immunocytochemical localization of GAD isoforms in the CNS ganglia of the cobweb spider,Achaearanea tepidariorum

Glutamic acid decarboxylase (GAD) is an enzyme that catalyzes the decarboxylation of glutamate to γ‐aminobutyric acid (GABA) and CO₂. It has been discovered that the GAD has a restricted tissue distribution and it is highly expressed in the cytoplasm of GABAergic neurons in the CNS where GABA is used as a neurotransmitter. We have examined the microstructure of ganglionic neurons and nerves arising from the CNS and describe here the immunocytochemical localization of GAD isoforms to reveal the ecophysiological significance of GABA for the web‐building spider's behavior. In the CNS of the cobweb spider, Achaearanea tepidariorum, immunocytochemical localization of GAD isoforms can be detected in the neurons and neuropiles of the optic lobes. In addition, GAD‐like immunoreactive cell bodies are observed at the intrinsic cell bodies near the central body and the symmetric cell clusters of the protocerebrum. However, the fibrous masses within the protocerebral ganglion are not labeled at all. Based on its interconnection with other regions of the CNS, our findings suggest that the central body in the web‐building spider may act as an association center as well as a visual center.     

Early embryonic development in the spider Achaearanea tepidariorum: Microinjection verifies that cellularization is complete before the blastoderm stage

The spider Achaearanea tepidariorum is emerging as a non-insect model for studying developmental biology. However, the availability of microinjection into early embryos of this spider has not been reported. We defined the early embryonic stages in A. tepidariorum and applied microinjection to its embryos. During the preblastoderm 16- and 32-nucleus stages, the energids were moving toward the egg periphery. When fluorochrome-conjugated dextran was microinjected into the peripheral region of 16-nucleus stage embryos, it was often incorporated into a single energid and inherited in the progeny without leaking out to surrounding energids. This suggested that 16-nucleus stage embryos consisted of compartments, each containing a single energid. These compartments were considered to be separate cells. Fluorochrome-conjugated dextran could be introduced into single cells of 16- to 128-nucleus stage embryos, allowing us to track cell fate and movement. Injection with mRNA encoding a nuclear localization signal/green fluorescent protein fusion construct demonstrated exogenous expression of the protein in live spider embryos. We propose that use of microinjection will facilitate studies of spider development. Furthermore, these data imply that in contrast to the Drosophila syncytial blastoderm embryo, the cell-based structure of the Achaearanea blastoderm embryo restricts diffusion of cytoplasmic gene products.     

Delta-Notch signaling is involved in the segregation of the three germ layers in Xenopus laevis

In vertebrates, the induction of the three germ layers (ectoderm, mesoderm and endoderm) has been extensively studied, but less is known about how they segregate. Here, we investigated whether Delta-Notch signaling is involved in this process. Activating the pathway in the marginal zone with Notch^ICD resulted in an expansion of endodermal and neural ectoderm precursors, leaving a thinner mesodermal ring around the blastopore at gastrula stage, when germ layers are segregated. On the other hand, when the pathway was blocked with Delta-1^STU or with an antisense morpholino oligonucleotide against Notch, the pan-mesodermal brachyury (bra) domain was expanded and the neural border was moved animalwards. Strikingly, the suprablastoporal endoderm was either expanded when Delta-1 signaling was blocked, or reduced after the general knock-down of Notch. In addition, either activating or blocking the pathway delays the blastopore closure. We conclude that the process of delimiting the three germ layers requires Notch signaling, which may be finely regulated by ligands and/or involve non-canonical components of the pathway. Moreover, Notch activity must be modulated at appropriate levels during this process in order to keep normal morphogenetic movements during gastrulation.     

Galphaq signaling is required for Rho-dependent transcriptional activation of the cyclooxygenase-2 promoter in fibroblasts

Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566). However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated. In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q). Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11). GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB). Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter. Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells. Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction. Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin. These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts.     

Delta-Notch signaling controls the generation of neurons/glia from neural stem cells in a stepwise process

We examined the role of Notch signaling on the generation of neurons and glia from neural stem cells by using neurospheres that are clonally derived from neural stem cells. Neurospheres prepared from Dll1(lacZ/lacZ) mutant embryos segregate more neurons at the expense of both oligodendrocytes and astrocytes. This mutant phenotype could be rescued when Dll1(lacZ/lacZ) spheres were grown and/or differentiated in the presence of conditioned medium from wild-type neurospheres. Temporal modulation of Notch by soluble forms of ligands indicates that Notch signaling acts in two steps. Initially, it inhibits the neuronal fate while promoting the glial cell fate. In a second step, Notch promotes the differentiation of astrocytes, while inhibiting the differentiation of both neurons and oligodendrocytes.     

Biomarker technology roundup: from discovery to clinical applications, a broad set of tools is required to translate from the lab to the clinic

Biomarkers are being utilized throughout the drug discovery and development process to understand fundamental biological processes and relationships. Specific biomarkers for disease states, prognosis, and response to therapy have been applied to screening tissues and serum, and serve as new tools in the development of therapeutics, to segment the population for specific treatments. The use of specific biomarkers to screen subjects to determine clinical trial eligibility, and for early toxicology studies, holds the potential to decrease drug failure rates in the later phases of the clinical trial process. Traditional research tools have been employed to study the genes, proteins, and metabolites of interest. In addition, new technologies and permutations of existing technologies have been developed particularly for investigation in the preclinical and clinical phases of drug development. More importantly, the transition of a compound from preclinical to clinical is aided by technologies that span both process segments. Identification of biomarkers that can be studied throughout the development process requires technologies that are both feasible and cost-effective for large patient populations.     

Zymogen activation in the streptokinase-plasminogen complex. Ile1 is required for the formation of a functional active site.

Plasminogen (Plgn) is usually activated by proteolysis of the Arg561-Val562 bond. The amino group of Val562 forms a salt-bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK.Plgn) which subsequently rearranges to an active complex (SK.Plgn*) although the Arg561-Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (muPlgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK.Plgn* by 100-fold (Ile1Val) to >/= 104-fold (Ile1-->Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK.Plgn but is necessary for SK.Plgn*. The substitution of Lys698 of muPlgn decreases the activity of SK.Plgn* by only 10-60-fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15-50-fold. These observations suggest that Lys698 is involved in formation of the initial SK.Plgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740.     

Laforin is required for the functional activation of malin in endoplasmic reticulum stress resistance in neuronal cells

Mutations in either EPM2A, the gene encoding a dual-specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease in humans. Lafora disease is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration and accumulation of poorly branched glycogen inclusions, called Lafora bodies or polyglucosan bodies, within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of Lafora disease remains unknown. Here, we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its Lafora disease-causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin-positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild-type malin nor its mutants elicit endoplasmic reticulum stress, although the mutants exaggerate the response to endoplasmic reticulum stress. Overexpressed laforin impairs the polyubiquitination of malin while it recruits malin to polyglucosan bodies. The recruitment and activities of laforin and malin are both required for the polyglucosan body disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin-malin complex plays a critical role in disrupting Lafora bodies and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in Lafora disease.     

Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function.     

Cysteine-rich region of X-Serrate-1 is required for activation of Notch signaling in Xenopus primary neurogenesis

The Notch family genes encode single-pass transmembrane proteins which function in a variety of cell fate specifications in invertebrates and vertebrates. In Xenopus primary neurogenesis, the Notch ligands, X-Delta-1 and X-Serrate-1, mediate Notch signaling and regulate cell differentiation. In the present study, we examined the role of the Serrate-specific cysteine-rich (CR) region in the primary neurogenesis of Xenopus embryos. The ligand constructs containing the DSL (Delta/Serrate/Lag-2) domain in the extracellular region caused a reduction in primary neurons, whereas the DSL-deleted form of X-Delta-1 resulted in the overproduction of primary neurons. However, the DSL-deleted form of X-Serrate-1 or the construct containing only the CR region in the extracellular domain (SerCR) reduced the number of primary neurons. In contrast, the CR-deleted form of X-Serrate-1 (SerACR) lost activity as a Notch ligand, regardless of the presence of the DSL domain within the extracellular domain. Overexpression of X-Delta-1 and X-Serrate-1 strongly induced the expression of Xenopus ESR-1 (XESR-1), a gene related to Drosophila Enhancer of split. SerCR alone also moderately induced the expression of XESR-1, but the SerACR form did not induce this expression. Co-injection of X-Notch-1deltaICD, which deletes the intracellular domain (ICD), with SerCR suppressed a neurogenic phenotype, although co-injection of X-Su(H)1DBM with SerCR did not, indicating that SerCR affects primary neurogenesis through the Notch/Su(H) pathway. These results suggestthatthe CR region of Xenopus Serrate is required for the activation of Notch signaling and cell fate specification in primary neurogenesis.     

Ethylene signaling is required for the acceleration of cell death induced by the activation of AtMEK5 in Arabidopsis

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of plant growth, development and responses to a wide variety of stimuli. In a conditional gain-of-function transgenic system, the activation of AtMEK5, a MAPK kinase, can in turn activate endogenous AtMAPK3 and AtMAPK6, and can lead to a striking increase in ethylene production and induce hypersensitive response (HR)-like cell death in Arabidopsis. However, the role of the increased ethylene production in regulating this HR-like cell death remains unknown. Using Arabidopsis transgenic plants that express AtMEK5(DD), an active mutant of AtMEK5 that is under the control of a steroid-inducible promoter, we tested the contribution of ethylene to cell death. We found that ethylene biosynthesis occurs before cell death. Cell death was delayed by inhibiting AtMEK5-induced ethylene production using inhibitors of ACC-synthases, ACC-oxidases or ethylene receptors. In the mutants AtMEK5(DD)/etr1-1 and AtMEK5(DD)/ein2-1, both of which showed insensitivity to ethylene, the expression of AtMEK5(DD) protein, activity of AtMAPK3 and AtMAPK6, and ethylene production were the same as those seen in AtMEK5(DD) transgenic plants, but cell death was also delayed. These data suggest that ethylene signaling perception is required to accelerate cell death that is induced by AtMEK5 activation.     

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration [1]. In bilaterians, there are at least 12 subfamilies of Wnt genes [2]. Wnt3 and Wnt8 are required for somitogenesis in vertebrates [3-7] and are thought to be involved in posterior specification in deuterostomes in general [8]. Although TCF and beta-catenin have been implicated in the posterior patterning of some short-germ insects [9, 10], the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. Here we investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum[11]. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. Our results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, we propose that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes.     

Phosphorylation of the activation loop tyrosines is required for sustained Syk signaling and growth factor-independent B-cell proliferation

The Syk kinase is regarded as a promising target for the treatment of antigen-driven B-cell malignancies, considering its essential role in propagating antigenic stimuli through the B-cell receptor (BCR). In certain common B-cell malignancies Syk is activated even in the absence of BCR engagement, suggesting a wider role for this kinase in lymphomagenesis. In this paper, we have profiled molecular differences between BCR-induced and constitutive Syk activation in terms of phosphorylation of regulatory tyrosine residues, downstream signaling properties and capacity to sustain B-cell proliferation. Analysis of primary chronic lymphocytic leukemia B-cells and diffuse large B-cell lymphoma cell lines revealed that constitutive and BCR-induced Syk activation differ with respect to the phosphorylation status of the regulatory tyrosines at positions 352 and 525/526, with only the first site being phosphorylated in the case of constitutive and both sites in the case of BCR-induced Syk activation. Syk phosphorylated only on Y352 is capable of downstream signaling, as evidenced by experiments with a phosphomimetic mutant in which the activation loop tyrosines (YY525/526) were replaced with phenylalanines. However, phosphorylation at YY525/526 was shown to significantly increase the enzymatic activity of Syk and to be required for sustained PLCγ2, Akt and ERK signaling as well as B-cell transformation. These data demonstrate that constitutively active Syk and Syk activated by BCR crosslinking represent separate stages of Syk activation with distinct signaling properties and transforming capacities.     

Parathyroid hormone receptor trafficking contributes to the activation of extracellular signal-regulated kinases but is not required for regulation of cAMP signaling

Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G(s)- and G(q)-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and beta-arrestin1 (beta-arrestin1-(319-418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1-34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg(11,13),Tyr36]PTHrP-(1-36)NH2), which selectively activates the G(s)/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G(q) (but not G(s)) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.     

HSP90 is required for TAK1 stability but not for its activation in the pro-inflammatory signaling pathway

The protein kinase transforming-growth-factor-β-activated kinase-1 (TAK1) is a key regulator in the pro-inflammatory signaling pathway and is activated by tumor necrosis factor-α, interleukin-1 (IL-1) and lipopolysaccharide (LPS). We describe the identification of TAK1 as a client protein of the 90 kDa heat-shock protein (Hsp90)/cell division cycle protein 37 (Cdc37) chaperones. However, Hsp90 is not required for the activation of TAK1 as short exposure to the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) did not affect its activation by LPS or IL-1. Prolonged treatment of cells with 17-AAG inhibits Hsp90 and downregulates TAK1. Our results suggest that Hsp90 is required for the folding and stability of TAK1 but is displaced and no longer required when TAK1 is complexed to TAK1-binding protein-1 (TAB1).

Structured summary

MINT-6797182:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with CDC37 (uniprotkb:Q16543) and HSP90 (uniprotkb:P07900) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797194:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB1 (uniprotkb:Q15750), HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797248:

TAK1 (uniprotkb:Q62073) physically interacts (MI:0218) with HSP90 (uniprotkb:P07901), CDC37 (uniprotkb:Q61081), TAB2 (uniprotkb:Q99K90) and TAB1 (uniprotkb:Q8CF89) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797232:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by pull down (MI:0096)

MINT-6797216:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB2 (uniprotkb:Q9NYJ8), CDC37 (uniprotkb:Q16543), HSP90 (uniprotkb:P07900) and TAB1 (uniprotkb:Q15750) by anti bait coimmunoprecipitation (MI:0006)

     

Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast

Signaling mucins are cell adhesion molecules that activate RAS/RHO guanosine triphosphatases and their effector mitogen-activated protein kinase (MAPK) pathways. We found that the Saccharomyces cerevisiae mucin Msb2p, which functions at the head of the Cdc42p-dependent MAPK pathway that controls filamentous growth, is processed into secreted and cell-associated forms. Cleavage of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p generated the active form of the protein by a mechanism incorporating cellular nutritional status. Activated Msb2p functioned through the tetraspan protein Sho1p to induce MAPK activation as well as cell polarization, which involved the Cdc42p guanine nucleotide exchange factor Cdc24p. We postulate that cleavage-dependent activation is a general feature of signaling mucins, which brings to light a novel regulatory aspect of this class of signaling adhesion molecule.     

XRad17 is required for the activation of XChk1 but not XCds1 during checkpoint signaling in Xenopus

The DNA damage/replication checkpoints act by sensing the presence of damaged DNA or stalled replication forks and initiate signaling pathways that arrest cell cycle progression. Here we report the cloning and characterization of Xenopus orthologues of the RFCand PCNA-related checkpoint proteins. XRad17 shares regions of homology with the five subunits of Replication factor C. XRad9, XRad1, and XHus1 (components of the 9-1-1 complex) all show homology to the DNA polymerase processivity factor PCNA. We demonstrate that these proteins associate with chromatin and are phosphorylated when replication is inhibited by aphidicolin. Phosphorylation of X9-1-1 is caffeine sensitive, but the chromatin association of XRad17 and the X9-1-1 complex after replication block is unaffected by caffeine. This suggests that the X9-1-1 complex can associate with chromatin independently of XAtm/XAtr activity. We further demonstrate that XRad17 is essential for the chromatin binding and checkpoint-dependent phosphorylation of X9-1-1 and for the activation of XChk1 when the replication checkpoint is induced by aphidicolin. XRad17 is not, however, required for the activation of XCds1 in response to dsDNA ends.     

Protein synthesis is a required process for the optic lobe circadian clock in the cricket Gryllus bimaculatus

The effects of a translation inhibitor, cycloheximide (CHX), on the circadian neuronal activity rhythm of the optic lamina-medulla compound eye complex cultured in vitro were investigated in the cricket Gryllus bimaculatus. When the complex was treated with 10(-5) M CHX for 6 h, the rhythm exhibited a marked phase shift. The magnitude and direction of the phase shift were dependent on the phase at which the complex was treated with CHX; phase delays occurred during the late subjective day to early subjective night, whereas phase advances occurred around the late subjective night. Continuous application of CHX abolished circadian rhythms of both the spontaneous neuronal activity and the visually evoked response. However, it abolished neither the spontaneous activity nor the visually evoked response. As washed with fresh medium after CHX treatment, the rhythm soon reappeared and the subsequent phase was clearly correlated to the termination time of the treatment. These results suggest that protein synthesis is also involved in the cricket optic lobe circadian clock, and that the clock-related protein synthesis may be active during the late subjective day to subjective night.