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Objective: To study enzymatic properties of intracellular maltogenic amylase (BMAL) in Bacillus sp. B110, purification, gene cloning, heterologous expression and purification were performed. It laid a foundation for later research on the development of new starch processing enzymes. Method: The full-length of BMAL gene was amplified by PCR and over-expressed and heterologous expressed in Escherichia coli. The recombinant enzyme was purified with nickel affinity chromatography (Ni2+-NTA), and its enzymatic characteristic were determined. The amino acid sequence of BMAL was analyzed by sequence analysis tools BioEdit and MEGA tools, and the three-dimensional model was predicted by alpha fold2. Results: The nucleotide sequence analysis revealed an open reading frame (ORF) of 1770 bp encoded a putative protein of 589 aa residues. The recombinant enzyme was purified with nickel affinity chromatography (Ni2+-NTA). SDS-PAGE analysis of the purified enzyme revealed that the molecular mass of BMAL was 63 kDa. The primary structure of BMAL was similar to those of MAases from B. subtilis 168 and B. subtilis SUH4-2, such as possession of an extra domain at its N-terminal and had a catalytic triad Asp328-Glu357-Asp424. The purified recombinant enzyme rBMAL had an optimum temperature of 45 ℃ and an optimum pH around 6.0. The rBMAL retained about 60% activity after 7 h of incubation at below 30 ℃, but it lost 98% of the original activity after 2 h of incubation at 60 ℃. These results revealed that the rBMAL was inthermostability. The activity of rBMAL was stable in pH7.0~9.5 at 4 ℃ for 12 h. In the presence of 1 mmol/L metal ion Mg2+, the activity of rBMAL increased by 36%, while 1 mmol/L Ni2+, Fe3+, Co2+, Cu2+, Zn2+, Al3+, Ca2+ inhibited the activity of rBMAL, which decreased by 85%~48%. The recombinant enzyme activity was inhibited by methanol, acetone, ethanol, acetonitrile, EDTA and SDS, and the enzyme activity was decreased to 32.3%~64.8%. Conclusion: The intracellular maltose amylase BMAL from Bacillus sp. B110 had high catalytic capacity and pH stability, which had potential application in bread baking industry.

We previously identified a critical pathogenic role for mineralo corticoid receptor (MR) activation in cardiomyocytes that included a potential interact ion between the MR and the molecular circadian clock. While glucocorticoid regulation of the circadian clock is undisputed, studies on MR interactions with circadian clock signalling are limited. We hypothesised that the MR influences cardiac circadian clock s ignalling, and vice versa. Aldosterone or corticosterone (10 nM) regulated Cry1, Per1, Per2 and ReverbA (Nr1d1) gene expression patterns in H9c2 cells over 24 h. MR-dependen t regulation of circadian gene promoters containing GREs and E-box sequences wa s established for CLOCK, Bmal, CRY1 and CRY2, PER1 and PER2 and transcriptional a ctivators CLOCK and Bmal modulated MR-dependent transcription of a subset of these promoters. We also demonstrated differential regulation of MR target gene expr ession in hearts of mice 4 h after administration of aldosterone at 08:00 h vs 20:0 0 h. Our data support MR regulation of a subset of circadian genes, with endogenous c ircadian transcription factors CLOCK and BMAL modulating the response. This unsuspecte d relationship links MR in the heart to circadian rhythmicity at the molecular level and has important implications for the biology of MR signalling in response to al dosterone as well as cortisol. These data are consistent with MR signalling in the b rain where, like the heart, it preferentially responds to cortisol. Given the undisputed requi rement for diurnal cortisol release in the entrainment of peripheral clocks, the present study highlights the MR as an important mechanism for transducing the circadian actions of cortisol in addition to glucocorticoid receptor (GR) in the heart. [ABSTRACT FROM AUTHOR]

Disregulation of genes making up the mammalian circadian clock has been associated with different forms of cancer. This study aimed to address how the circadian clock genes behave over the course of treatment for both the acute and chronic forms of leukemia and whether any could be used as potential biomarkers as a read-out for therapeutic efficacy. Expression profiling for both core and ancillary clock genes revealed that the majority of clock genes are down-regulated in acute myeloid leukemia patients, except for Cry2, which is up-regulated towards the end of treatment. A similar process was seen in acute lymphocytic leukemia patients; however, here, Cry2 expression came back up towards control levels upon treatment completion. In addition, all of the core clock genes were down-regulated in both chronic forms of leukemia (chronic myeloid leukemia and chronic lymphocytic leukemia), except for Cry2, which was not affected when the disease was diagnosed. Furthermore, the NAD(+) – dependent protein deacetylase Sirt1 has been proposed to have a dual role in both control of circadian clock circuitry and promotion of cell survival by inhibiting apoptotic pathways in cancer. We used a pharmacological-based approach to see whether Sirt1 played a role in regulating the circadian clock circuitry in both acute and chronic forms of leukemia. Our results suggest that interfering with Sirt1 leads to a partial restoration of BMAL1 oscillation in chronic myeloid leukemia patient samples. Furthermore, interfering with Sirt1 activity led to both the induction and repression of circadian clock genes in both acute and chronic forms of leukemia, which makes it a potential therapeutic target to either augment existing therapies for chronic leukemia or to act as a means of facilitating chronotherapy in order to maximize both the effectiveness of existing therapies and to minimize therapy-associated toxicity.

Disregulation of genes making up the mammalian circadian clock has been associated with different forms of cancer. This study aimed to address how the circadian clock genes behave over the course of treatment for both the acute and chronic forms of leukemia and whether any could be used as potential biomarkers as a read-out for therapeutic efficacy. Expression profiling for both core and ancillary clock genes revealed that the majority of clock genes are down-regulated in acute myeloid leukemia patients, except for Cry2, which is up-regulated towards the end of treatment. A similar process was seen in acute lymphocytic leukemia patients; however, here, Cry2 expression came back up towards control levels upon treatment completion. In addition, all of the core clock genes were down-regulated in both chronic forms of leukemia (chronic myeloid leukemia and chronic lymphocytic leukemia), except for Cry2, which was not affected when the disease was diagnosed. Furthermore, the NAD(+) -- dependent protein deacetylase Sirt1 has been proposed to have a dual role in both control of circadian clock circuitry and promotion of cell survival by inhibiting apoptotic pathways in cancer. We used a pharmacological-based approach to see whether Sirt1 played a role in regulating the circadian clock circuitry in both acute and chronic forms of leukemia. Our results suggest that interfering with Sirt1 leads to a partial restoration of BMAL1 oscillation in chronic myeloid leukemia patient samples. Furthermore, interfering with Sirt1 activity led to both the induction and repression of circadian clock genes in both acute and chronic forms of leukemia, which makes it a potential therapeutic target to either augment existing therapies for chronic leukemia or to act as a means of facilitating chronotherapy in order to maximize both the effectiveness of existing therapies and to minimize therapy-associated toxicity. [ABSTRACT FROM AUTHOR]

Borderline personality disorder (BPD) is characterised by a deep-reaching pattern of affective instability, incoherent identity, self-injury, suicide attempts, and disturbed interpersonal relations and lifestyle. The daily activities of BPD patients are often chaotic and disorganized, with patients often staying up late while sleeping during the day. These behavioural patterns suggest that altered circadian rhythms may be associated with BPD. Furthermore, BPD patients frequently report suffering from sleep disturbances. In this review, we overview the evidence that circadian rhythms and sleep are disturbed in BPD, and we explore the possibility that personality traits that are pertinent for BPD may be associated with circadian typology, and perhaps to circadian genotypes. With regards to sleep architecture, we review the evidence that BPD patients display altered non-REM and REM sleep. A possible cue to a deeper understanding of this temporal dysregulation might be an analysis of the circadian clock at the molecular and cellular level, as well as behavioural studies using actigraphy and we suggest avenues for further exploration of these factors. [ABSTRACT FROM AUTHOR]

To identify circadian patterns of gene expression in skeletal muscle, adult male zebrafish were acclimated for 2 wk to a 12:12-h light-dark photoperiod and then exposed to continuous darkness for 86 h with ad libitum feeding. The increase in gut food content associated with the subjective light period was much diminished by the third cycle, enabling feeding and circadian rhythms to be distinguished. Expression of zebrafish paralogs of mammalian transcriptional activators of the circadian mechanism (bmal1, clock1, and rora) followed a rhythmic pattern with a ~24-h periodicity. Peak expression of rora paralogs occurred at the beginning of the subjective light period [Zeitgeber time (ZT)07 and ZT02 for roraa and rorab], whereas the highest expression of bmal1 and clock paralogs occurred 12 h later (ZT13-15 and ZT16 for bmal and clock paralogs). Expression of the transcriptional repressors cry1a, per1a/1b, per2, per3, nr1d2a/2b, and nr1d1 also followed a circadian pattern with peak expression at ZT0-02. Expression of the two paralogs of cry2 occurred in phase with clock1a/1b. Duplicated genes had a high correlation of expression except for paralogs of clock1, nr1d2, and per1, with cry1b showing no circadian pattern. The highest expression difference was 9.2-fold for the activator bmal1b and 51.7-fold for the repressor per1a. Out of 32 candidate clock-controlled genes, only myf6, igfbp3, igfbp5b, and hsf2 showed circadian expression patterns. Igfbp3, igfbp5b, and myf6 were expressed in phase with clock1a/1b and had an average of twofold change in expression from peak to trough, whereas hsf2 transcripts were expressed in phase with cry1a and had a 7.2-fold-change in expression. The changes in expression of clock and clock-controlled genes observed during continuous darkness were also observed at similar ZTs in fish exposed to a normal photoperiod in a separate control experiment. The role of circadian clocks in regulating muscle maintenance and growth are discussed. [ABSTRACT FROM AUTHOR]

On the first day of development a circadian clock becomes functional in the zebrafish embryo. How this oscillator is set in motion remains unclear. We demonstrate that zygotic period1 transcription begins independent of light exposure. Pooled embryos maintained in darkness and under constant temperature show elevated non-oscillating levels of period1 expression. Consequently, there is no maternal effect or developmental event that sets the phase of the circadian clock. Analysis of period1 transcription, at the cellular level in the absence of environmental stimuli, reveals oscillations in cells that are asynchronous within the embryo. Demonstrating an autonomous onset to rhythmic period1 expression. Transcription of clock1 and bmal1 is rhythmic in the adult, but constant during development in light-entrained embryos. Transient expression of dominant-negative ΔCLOCK blocks period1 transcription, thus showing that endogenous CLOCK is essential for the transcriptional regulation of period1 in the embryo. We demonstrate a default mechanism in the embryo that initiates the autonomous onset of the circadian clock. This embryonic clock is differentially regulated from that in the adult, the transition coinciding with the appearance of several clock output processes. [ABSTRACT FROM AUTHOR]

The section presents abstracts on medical topics which include the societal burden of untreated sleep apnea, polysomnographic patterns related to the main risk factors of childhood obstructive sleep apnea syndrome, and the epidemic of obesity in children.

The daily biological clock regulates the timing of sleep and physiological processes that are of fundamental importance to human health, performance, and well-being. Environmental parameters of relevance to biological clocks include (1) daily fluctuations in light intensity and temperature, and (2) seasonal changes in photoperiod (day length) and temperature; these parameters vary dramatically as a function of latitude and locale. In wide-ranging species other than humans, natural selection has genetically optimized adaptiveness along latitudinal clines. Is there evidence for selection of clock gene alleles along latitudinal/photoperiod clines in humans? Anumber of polymorphisms in the human clock genes Per2, Per3, Clock, and AANAT have been reported as alleles that could be subject to selection. In addition, this investigation discovered several novel polymorphisms in the human Arntl and Arntl2 genes that may have functional impact upon the expression of these clock transcriptional factors. The frequency distribution of these clock gene polymorphisms is reported for diverse populations of African Americans, European Americans, Ghanaians, Han Chinese, and Papua New Guineans (including 5 subpopulations within Papua New Guinea). There are significant differences in the frequency distribution of clock gene alleles among these populations. Population genetic analyses indicate that these differences are likely to arise from genetic drift rather than from natural selection. [ABSTRACT FROM AUTHOR]

Pineal serotonin- N-acetyltransferase (arylalkylamine- N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat ( aanat2) of the gilthead seabream ( Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: The interaction between the BMAL1/CLOCK transcription factor and the cis-acting element known as the E-Box is a key event in the regulation of clock and clock-controlled gene expression. However, the fact that the ubiquitous E-Box element sits at the center of a presumably highly discriminating control system generates a certain level of puzzlement. Widely spread E-Boxes with a generic sequence CANNTG have been associated with expression of genes involved in a host of disparate biological processes, including the orchestration of circadian physiology. The intriguing specificity of this short DNA consensus element begs the hypothesis that its actual circadian properties might be encoded elsewhere, e.g., other factors or adjacent sequences. In a previous study, we found evidence that a short sequence in the mouse arginine vasopressin (AVP) proximal promoter has the ability to confer robust BMAL1/CLOCK responsiveness onto an adjacent E-Box. Here, we report the systematic analysis of this element. Our findings further define the determining features and sequence boundaries of this element, establish the effect of the photoperiod upon its interacting protein(s), and suggest that its cognate binding activity might be modulated by Zn2+ in a peripheral oscillator. [Copyright &y& Elsevier]

Circadian variation in plasminogen activator inhibitor-1 (PAI-1) production likely contributes to increased risk of myocardial infarction and decreased efficacy of thrombolytic therapy during the morning. In this study, we characterize the abilities of fundamental molecular components of intrinsic circadian clocks to regulate the human PAI-1 promoter in transfected endothelial cells. Both CLOCK:BMAL1 and CLOCK:BMAL2 heterodimers activate the PAI-1 promoter through requisite proximal (–565 to –560 bp) and distal (–680 to –675 bp) E-box enhancers. Although the distal E-box overlaps the 4G/5G polymorphism of the PAI-1 promoter, allelic variation at this site does not influence CLOCK:BMAL1-and CLOCK:BMAL2-mediated transactivation. Together, CLOCK:BMAL1 and CLOCK:BMAL2 make additive contributions to PAI-1 gene transcription. While the abilities of these heterodimers to activate gene expression differ by twofold, the susceptibilities of these circadian activators to inhibition by period and cryptochrome proteins are equivalent and redox independent. Given that BMAL1 and BMAL2 differ in their spatiotemporal distributions, such distinctions may allow intrinsic circadian clocks to modulate the amplitudes of their oscillators, while maintaining circadian periodicity. In this way, fundamental circadian clock components may drive circadian variation in PAI-1, which in turn influences the pathogenesis, timing, and treatment of acute atherothrombotic events. [Copyright &y& Elsevier]

The bHLH-PAS (basic helix-loop-helix/ Period-ARNT-Single minded) proteins are a family of transcriptional regulators commonly occurring in living organisms. bHLH-PAS members act as intracellular and extracellular "signals" sensors, initiating response to endo- and exogenous signals, including toxins, redox potential, and light. The activity of these proteins as transcription factors depends on nucleocytoplasmic shuttling: the signal received in the cytoplasm has to be transduced, via translocation, to the nucleus. It leads to the activation of transcription of particular genes and determines the cell response to different stimuli. In this review, we aim to present the current state of knowledge concerning signals that affect shuttling of bHLH-PAS transcription factors. We summarize experimentally verified and published nuclear localization signals/nuclear export signals (NLSs/NESs) in the context of performed in silico predictions. We have used most of the available NLS/NES predictors. Importantly, all our results confirm the existence of a complex system responsible for protein localization regulation that involves many localization signals, which activity has to be precisely controlled. We conclude that the current stage of knowledge in this area is still not complete and for most of bHLH-PAS proteins an experimental verification of the activity of further NLS/NES is needed. [ABSTRACT FROM AUTHOR]

Background: In addition to cognitive decline, Alzheimer's Disease (AD) is also characterized by agitation and disruptions in activity and sleep. These symptoms typically occur in the evening or night and have been referred to as 'sundowning'. They are especially difficult for carers and there are no specific drug treatments. There is increasing evidence that these symptoms reflect pathology of circadian rhythm generation and transmission., Objective: We investigated whether a transgenic mouse model relevant to AD (APPswe/PS1dE9) exhibits circadian alterations in locomotor activity in their home cage and whether expression of clock genes involved in the regulation of the circadian cycle is abnormal in the hippocampus and medulla-pons brain regions isolated from these mice., Results: In 2month old female mice the APPswe/PS1dE9 transgene alters levels and patterns in circadian rhythm of locomotor activity. Expression of the clock genes Per1, Per2, Cry1 and Cry2 was found to increase at night compared to day in wild-type control mice in the medulla/pons. This effect was blunted for Cry1 and Cry2 gene expression in APPswe/PS1dE9., Conclusion: This study suggests altered circadian regulation of locomotor activity is abnormal in female APPswe/ PS1dE9 mice and that this alteration has biomolecular analogies in a widely available model of AD. The early age at which these effects are manifest suggests that these circadian effects may precede plaque development. The APPswe/PS1dE9 mouse genetic model may have potential to serve as a tool in understanding the neuropathology of circadian abnormalities in AD and as a model system to test novel therapeutic agents for these symptoms., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)