Preparation and Characterization of an Anti-DNA Monoclonal Antibody Showing Size Selectivity Toward DNA Fragments

Onishi, Yoshiaki; Kato, Megumi; Hanyu, Yoshiro

doi:10.1089/hyb.2004.23.311

Cited by 4 publications

(3 citation statements)

References 27 publications

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“…SDS/PAGE and Western blotting proceeded as described previously [24]. Briefly, proteins were resolved by SDS/PAGE (9% gel) and transferred to PVDF membranes (Amersham).…”

Section: Western Blottingmentioning

confidence: 99%

The harmala alkaloid harmine is a modulator of circadian Bmal1 transcription

et al. 2011

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Biological rhythms are orchestrated by a cell-autonomous clock system that drives the rhythmic cascade of clock genes. We established an assay system using NIH 3T3 cells stably expressing the Bmal1 promoter-driven luciferase reporter gene and used it to analyse circadian oscillation of the gene. Modulators of PKC (protein kinase C) revealed that an activator and an inhibitor represented short- and long-period phenotypes respectively which were consistent with reported effects of PKC on the circadian clock and validated the assay system. We examined the effects of the alkaloid harmine, contained in Hoasca, which has a wide spectrum of pharmacological actions, on circadian rhythms using the validated assay system. Harmine dose dependently elongated the period. Furthermore, EMSA (electrophoretic mobility-shift assay) and Western-blot analysis showed that harmine enhanced the transactivating function of RORα (retinoid-related orphan receptor α), probably by increasing its nuclear translocation. Exogenous expression of RORα also caused a long period, confirming the phenotype indicated by harmine. These results suggest that harmine extends the circadian period by enhancing RORα function and that harmine is a new candidate that contributes to the control of period length in mammalian cells.

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“…SDS/PAGE and Western blotting proceeded as described previously [24]. Briefly, proteins were resolved by SDS/PAGE (9% gel) and transferred to PVDF membranes (Amersham).…”

Section: Western Blottingmentioning

confidence: 99%

The harmala alkaloid harmine is a modulator of circadian Bmal1 transcription

et al. 2011

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“…SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting were performed as previously described (27). Proteins were resolved by 7% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham).…”

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confidence: 99%

Rhythmic SAF-A Binding Underlies Circadian Transcription of the Bmal1 Gene

Onishi

Hanai

Ohno

et al. 2008

Molecular and Cellular Biology

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Although Bmal1 is a key component of the mammalian clock system, little is understood about the actual mechanism of circadian Bmal1 gene transcription, particularly at the chromatin level. Here we discovered a unique chromatin structure within the Bmal1 promoter. The RORE region, which is a critical cis element for the circadian regulation of the Bmal1 gene, is comprised of GC-rich open chromatin. The 3-flanking region of the promoter inhibited rhythmic transcription in the reporter gene assay in vitro even in the presence of ROR␣ and REV-ERB␣. We also found that the nuclear matrix protein SAF-A binds to the 3-flanking region with circadian timing, which was correlated with Bmal1 expression by footprinting in vivo. These results suggest that the unique chromatin structure containing SAF-A is required for the circadian transcriptional regulation of the Bmal1 gene in cells.Circadian rhythms in behavior and physiology have an adaptive significance for living organisms from bacteria to humans and reflect the existence of an underlying intrinsic circadian oscillator or biological clock (10). The master clock that generates circadian rhythms in mammals is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. In turn, peripheral clocks directly regulate many local rhythms that probably feed back to the SCN through hypothalamic integration (5). The molecular mechanism of the circadian oscillator consists of autoregulatory transcriptional and translational feedback loops that have both positive and negative elements (10). The key transcription factors, CLOCK and BMAL1, form heterodimers that bind to E-box enhancer sequences and activate the transcription of the three Period genes (Per1, Per2, and Per3) and two Cryptochrome genes (Cry1 and Cry2) (46). The PER and CRY proteins subsequently repress transcription at their own promoters through negative feedback caused by acting on the CLOCK-BMAL1 complex (35). This feedback loop system controls the central clock in the SCN and the peripheral clocks in most peripheral tissues (18).Originally, BMAL1 (also known as MOP3) was characterized by high expression levels in brain and muscle cells (16). As the activity of Bmal1 Ϫ/Ϫ mice immediately becomes completely arrhythmic in constant darkness, BMAL1 is apparently an essential and nonredundant component of the mammalian clock (6). The level of Bmal1 transcripts robustly oscillates in the SCN and in peripheral clock cells (25), and the circadian regulation of Bmal1 transcription contributes to the formation of interconnected feedback loops (35). The Bmal1 promoter contains two recognition motifs for ROR and REV-ERB orphan nuclear receptors (ROREs). Preitner et al. reported that REV-ERB␣, which represses Bmal1 expression, is the major regulator of cyclic Bmal1 transcription (31), and Akashi and Takumi described that ROR␣ acts to promote Bmal1 transcription (1). The opposing activities of the orphan nuclear receptors ROR␣ and REV-ERB␣ are important in the maintenance of circadian clock function (34). It was also suggested...

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“…Using this cDNA as the template, the heavychain variable region (V H ) and the light-chain variable region (V L ) were amplified by PCR, and their nucleotide sequences were determined [22]. Corresponding cDNA was synthesized.…”

Section: Sequence Analysis In Cdr Regions Of the Functional Monoclonamentioning

confidence: 99%

Inhibition of Gaussia luciferase Activity by a Monoclonal Antibody

Kato

Chiba

Hanyu

2012

CEI

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A new electrofusion method has been developed with high efficiency for hybridoma formation. This method induces more positive clones than conventional fusion methods do. This has led to the successful establishment of functional monoclonal antibodies that can modulate the function of antigens. Such antibodies are difficult to obtain using existing methods. Monoclonal antibodies that inhibit the luminescent activity of the Gaussia luciferase (Gluc) have been prepared using this method. The affinities of antibodies for their antigens were assessed qualitatively by Biacore measurements. Epitope mapping experiments showed that these antibodies recognize the conformation of Gluc antigens rather than their specific sequences. The amino acid sequence of an antigen-recognition region of an antibody was also determined. The mechanism of luminescence inhibition by the monoclonal antibodies is discussed.

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Preparation and Characterization of an Anti-DNA Monoclonal Antibody Showing Size Selectivity Toward DNA Fragments

Cited by 4 publications

References 27 publications

The harmala alkaloid harmine is a modulator of circadian Bmal1 transcription

The harmala alkaloid harmine is a modulator of circadian Bmal1 transcription

Rhythmic SAF-A Binding Underlies Circadian Transcription of the Bmal1 Gene

Inhibition of Gaussia luciferase Activity by a Monoclonal Antibody

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