Histochemical Imaging of Alkaline Phosphatase Using a Novel Fluorescent Substrate

Takahashi, Toshifumi; Otsubo, Tadamune; Ikeda, Kiyoshi; Minami, Akira; Suzuki, Takashi

doi:10.1248/bpb.b14-00456

Cited by 9 publications

(5 citation statements)

References 27 publications

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“…Blocking the hydroxy group by phosphorylation, as shown for III-IX, can trap HBT unit in the enol form and prohibit the ESIPT process. (Figure 3) [29,32,34,35]. E matic cleavage of the phosphoryl group unveils the hydroxy group, and the HBT uni undergo ESIPT with accompanying emission, typically in the green region (Figure 4) Blocking the hydroxy group by phosphorylation, as shown for III-IX, can trap the HBT unit in the enol form and prohibit the ESIPT process.…”

Section: Reconnaissancementioning

confidence: 99%

“…E matic cleavage of the phosphoryl group unveils the hydroxy group, and the HBT uni undergo ESIPT with accompanying emission, typically in the green region (Figure 4) Blocking the hydroxy group by phosphorylation, as shown for III-IX, can trap the HBT unit in the enol form and prohibit the ESIPT process. (Figure 3) [29,32,34,35]. Enzymatic cleavage of the phosphoryl group unveils the hydroxy group, and the HBT unit can undergo ESIPT with accompanying emission, typically in the green region (Figure 4).…”

Section: Reconnaissancementioning

confidence: 99%

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Tailoring the AIE Chromogen 2-(2-Hydroxyphenyl)benzothiazole for Use in Enzyme-Triggered Molecular Brachytherapy

Dou

Nguyen

et al. 2022

Molecules

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A targeted strategy for treating cancer is antibody-directed enzyme prodrug therapy, where the enzyme attached to the antibody causes conversion of an inactive small-molecule prodrug into an active drug. A limitation may be the diffusion of the active drug away from the antibody target site. A related strategy with radiotherapeutics entails enzymatically promoted conversion of a soluble to insoluble radiotherapeutic agent, thereby immobilizing the latter at the target site. Such a molecular brachytherapy has been scarcely investigated. In distinct research, the advent of molecular designs for aggregation-induced emission (AIE) suggests translational use in molecular brachytherapy. Here, several 2-(2-hydroxyphenyl)benzothiazole substrates that readily aggregate in aqueous solution (and afford AIE) were elaborated in this regard. In particular, (1) the 2-(2-hydroxyphenyl) unit was derivatized to bear a pegylated phosphodiester that imparts water solubility yet undergoes enzymatic cleavage, and (2) a p-phenol unit was attached to the benzo moiety to provide a reactive site for final-step iodination (here examined with natural abundance iodide). The pegylated phosphodiester-iodinated benzothiazole undergoes conversion from aqueous-soluble to aqueous-insoluble upon treatment with a phosphatase or phosphodiesterase. The aggregation is essential to molecular brachytherapy, whereas the induced emission of AIE is not essential but provides a convenient basis for research development. Altogether, 21 compounds were synthesized (18 new, 3 known via new routes). Taken together, blending biomedical strategies of enzyme prodrug therapy with materials chemistry concerning substances that undergo AIE may comprise a step forward on the long road toward molecular brachytherapy.

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Section: Reconnaissancementioning

confidence: 99%

Section: Reconnaissancementioning

confidence: 99%

Tailoring the AIE Chromogen 2-(2-Hydroxyphenyl)benzothiazole for Use in Enzyme-Triggered Molecular Brachytherapy

Dou

Nguyen

et al. 2022

Molecules

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“…By using fluorescence imaging of sialidase activity by BTP3-Neu5Ac, detection of sialidase-bearing viruses and cells infected with viruses including influenza virus and visualization of sialidase activity using mammalian tissue sections are possible (13). Besides sialidase, β-galactosidase and alkaline phosphatase can also be fluorescently imaged by galactose or phosphate-conjugated BTP3 (14)(15).…”

Section: B Sialidase Fluorescent Imaging Probe Btp3-neu5acmentioning

confidence: 99%

Detection and Isolation of a Drug-Resistant Influenza Virus Using a Sialidase Fluorescence Imaging Technique

Kurebayashi

Takahashi

Otsubo

et al. 2020

TIGG

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Neuraminidase (NA), which is expressed on the membrane surface of an influenza virus, plays a critical role in the growth of a virus exhibiting sialidase activity, and this activity is a target for therapeutic drugs. The authors have developed a novel substrate, BTP3-Neu5Ac, that is capable of fluorescence imaging of sialidase activity and have developed a method for detection of influenza virus by fluorescence imaging of sialidase activity. The method has been applied to the detection and isolation of a drug-resistant influenza virus. Water-soluble BTP3-Neu5Ac undergoes hydrolysis by sialidase resulting in the liberation of sialic acid and generation of free BTP3. BTP3 is deposited and enabling fluorescence imaging of sialidase activity. Fluorescence imaging of the sialidase activity of influenza virus NA enables convenient and rapid detection of viruses and infected cells. A drug-resistant influenza virus, which has become a problem in recent years, is a virus that has acquired by mutation resistance to a sialidase inhibitor and retains its activity even in the presence of a sialidase inhibitor. The authors have therefore developed a method for selective detection of a drugresistant influenza virus. BTP3-Neu5Ac for detection and isolation of a drug-resistant influenza virus is expected to be widely used in the future as a useful tool in basic research, hygiene and clinical fields.

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Section: )mentioning

confidence: 99%

“…The water-soluble non-fluorescent BTP3-Neu5Ac is cleaved to a water-insoluble fluorescent benzothiazolylphenol derivative (BTP3) 6,7) and N-acetylneuraminic acid (Neu5Ac) by sialidase activity. 8) BTP3 locally deposits on sialidase activity-existing sites, resulting in local and fluorescent visualization of sialidase activity in live cells (Fig.…”

mentioning

confidence: 99%

Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses

Takahashi

Takano

Kurebayashi

et al. 2015

Biological & Pharmaceutical Bulletin

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Human parainfluenza virus type 1 (hPIV1) does not form clear plaque by the conventional plaque formation assay because of slightly a cytopathic effects in many cell lines infected with hPIV1, thus making in virus titration, isolation and inhibitor evaluation difficult. We have succeeded in fluorescent histochemical visualization of sialidase activities of influenza A and B viruses, Newcastle disease virus and Sendai virus by using a novel fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). In this study, we applied the BTP3-Neu5Ac assay for rapid detection of hPIV1 and hPIV type 3. The BTP3-Neu5Ac assay could histochemically visualize dot-blotted hPIVs on a membrane and hPIV-infected cells as local fluorescence under UV irradiation. We succeeded in distinct fluorescent visualization of hPIV1-infected cells in only 3 d using the BTP3-Neu5Ac assay. Due to there being no fixation, hPIV1 was isolated directly from fluorescent stained focus cells by the BTP3-Neu5Ac assay. Establishment of a sensitive, easy, and rapid fluorescent focus detection assay for hPIV, hPIV1 in particular will contribute greatly to progress in hPIV studies.Key words human parainfluenza virus (hPIV); sialidase activity; fluorescent visualization; histochemical staining;Human parainfluenza virus type 1 (hPIV1) and human parainfluenza virus type 3 (hPIV3) belong to genus Respirovirus, subfamily Paramyxovirinae, and family Paramyxoviridae. The hPIVs cause flu-like respiratory symptoms, including croup, pneumonia and bronchiolitis, mainly in infants younger than 5 years old and immunocompromised patients. Severe symptoms often lead to hospitalization and occasionally result in sudden death of newborns and infants. hPIVs, the majority of which are hPIV1 and hPIV3, are detected from approximately 20% of viral pneumonia cases in infants younger than 5 years of age.1,2) Although hPIVs are clinically important respiratory viruses for infants and immunocompromised patients, vaccines and medicines against hPIVs have not been developed.Clinical samples and virus culture supernatants include some viruses and mutants. When viruses replicate in the presence of a certain inhibitor, inhibitor-resistant mutant viruses may appear in the culture supernatant. To separate a virus isolate from such samples, the plaque formation assay is frequently used. A virus infects a cell and produces progeny viruses that spread to infect neighboring cells. The population of infected cells shows a cytopathic effect (CPE), for example, cell death or syncytium formation. When virus-cultured cells are overlaid by a medium containing agarose, cell lysis induced by CPE is visualized as a plaque. A virus isolate can be picked up directly from a plaque, which is formed by an isolate. hPIV3 has been reported to form large plaques in human epidermoid cancer (HEp-2) cells, in which hPIV3 shows much syncytium formation.3) On the other hand, hPIV1 does not form plaques by the normal plaq...

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Histochemical Imaging of Alkaline Phosphatase Using a Novel Fluorescent Substrate

Cited by 9 publications

References 27 publications

Tailoring the AIE Chromogen 2-(2-Hydroxyphenyl)benzothiazole for Use in Enzyme-Triggered Molecular Brachytherapy

Tailoring the AIE Chromogen 2-(2-Hydroxyphenyl)benzothiazole for Use in Enzyme-Triggered Molecular Brachytherapy

Detection and Isolation of a Drug-Resistant Influenza Virus Using a Sialidase Fluorescence Imaging Technique

Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses

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