Arsenic Compounds Accumulated in Sedimentary Microorganisms Cultivated in Media Containing Several Arsenicals

Sediments, as sources of microorganisms, were added to two kinds of media, 1/5 ZoBell 2216E and a solution of inorganic salts, which contained inorganic arsenic(III), inorganic arsenic(V), methanearsonic acid, dimethyl- arsinic acid, trimethylarsine oxide, tetramethylarsonium salt or arsenocholine. After 17 days of incubation at 20 °C, the arsenicals that had accumulated in the microorganisms were analysed by high-performance liquid chromatography (HPLC). While the more toxic arsenicals [inorganic arsenic(III), inorganic arsenic(V), methanearsonic acid, dimethylarsinic acid] were not converted in the microorganisms, trimethylarsine oxide and tetramethylarsonium salt were considerably degraded to inorganic arsenic(V), and arsenocholine to arsenobetaine. Arsenobetaine that had accumulated in the microorganisms was extracted and confirmed by thin-layer chromatography (TLC) and fast atom bombardment (FAB) mass spectrometry.     

Preparation of mesoporous silicas using food grade emulsifiers and its application for enzyme supports

A novel mesoporous silica (TMPS) was synthesized via self-assembly using a myristic acid ester of pentaglycerol. The ester is obtained from catalytic esterification and it is commercially available as a food grade emulsifier. TMPS material was employed for preparation of a biocatalyst in order to examine the ability as an enzyme support in comparison with the other mesoporous silica materials having a channel or a cage-like pore system. The used TMPS materials possessed the interconnected channel-like pore system with the pore sizes of 9.2, 12, and 16 nm. The materials successfully entrapped lipase into their mesopores with the high loadings. The resultant lipase/TMPS conjugates functioned as the biocatalyst for hydrolysis of p-nitrophenyl propionate (p-NPP), having the higher activity than those of the used mesoporous silica conjugates. The high activities were ascribed to the textural properties such as the small particle length, large pore size and the three-dimensional pore connectivity that permit the accessibility of p-NPP to the immobilized lipases during the reactions. Consequently, we concluded that TMPS materials are of the suitable mesoporous support for the enzymes.     

Design strategy for a near-infrared fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron dipyrromethene

Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a F?rster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.     

Development of a fluorescein analogue, TokyoMagenta, as a novel scaffold for fluorescence probes in red region

We present a design strategy for fluorescence probes with a high off/on activation ratio in the red wavelength region, based on a novel fluorescein analogue in which the O atom at the 10 position of the xanthene chromophore is replaced with a Si atom. To demonstrate the usefulness of this strategy, we designed and synthesized a red-fluorescent probe for β-galactosidase, and showed that it works in live HEK293 cells.     

Development of a far-red to near-infrared fluorescence probe for calcium ion and its application to multicolor neuronal imaging

To improve optical imaging of Ca(2+) and to make available a distinct color window for multicolor imaging, we designed and synthesized CaSiR-1, a far-red to near-infrared fluorescence probe for Ca(2+), using Si-rhodamine (SiR) as the fluorophore and the well-known Ca(2+) chelator BAPTA. This wavelength region is advantageous, affording higher tissue penetration, lower background autofluorescence, and lower phototoxicity in comparison with the UV to visible range. CaSiR-1 has a high fluorescence off/on ratio of over 1000. We demonstrate its usefulness for multicolor fluorescence imaging of action potentials (visualized as increases in intracellular Ca(2+)) in brain slices loaded with sulforhodamine 101 (red color; specific for astrocytes) that were prepared from transgenic mice in which some neurons expressed green fluorescent protein.     

Selective two-step labeling of proteins with an off/on fluorescent probe

We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis(6)-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His(6)Cys-tagged enhanced blue fluorescent protein (EBFP), but not His(6)-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.     

Rational Design of a Near‐infrared Fluorescence Probe for Ca2+ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Fluorescence imaging in the near‐infrared (NIR) region (650–900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)‐mediated fluorescence quenching of silicon‐ and phosphorus‐substituted rhodamines (SiRs and PRs) in order to guide the development of improved far‐red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca²⁺, CaPR‐1 , and its membrane‐permeable acetoxymethyl derivative, CaPR‐1 AM , which is distributed to the cytosol, in marked contrast to our previously reported Ca²⁺ far‐red to NIR fluorescence probe based on the SiR scaffold, CaSiR‐1 AM , which is mainly localized in lysosomes as well as cytosol in living cells. CaPR‐1 showed longer‐wavelength absorption and emission (up to 712 nm) than CaSiR‐1 . The new probe was able to image Ca²⁺ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.     

Distribution of inorganic arsenic and methylated arsenic in marine organisms

Inorganic arsenic and methylated arsenic compounds in 60 specimens of marine organisms were investigated by hydride generation derivatization and cold-trap gas chromatography–mass spectrometry (GC MS). Chloroform–methanol extracts from seaweeds, shellfish, fish, crustaceans and other marine organisms were separated into water-soluble and lipid-soluble fractions. The arsenic compounds in each fraction were identified and analysed as arsine, methylarsine, dimethylarsine and trimethylarsine. Trimethylarsenic compounds were distributed mainly in the water-soluble fraction of muscle of carnivorous gastropods, crustaceans and fish. The amounts of dimethylated arsenic compounds were found to be larger than that of trimethylated arsenic in the lipid-soluble fraction of fish viscera. Dimethylated arsenic compounds were distributed in the water-soluble fraction of Phaeophyceae.