21 research outputs found
One-Pot Synthesis of Highly Dispersible Fluorescent Nanodiamonds for Bioconjugation
Fluorescent
nanodiamonds (FNDs) have been attracting much attention
as promising therapeutic agents and probes for bioimaging and nanosensing.
For their biological applications, several hydrophilizing methods
to enhance FND colloidal stability have been developed to suppress
their aggregation and the nonspecific adsorption to biomolecules in
complex biomedical environments. However, these methods involve several
complicated synthetic and purification steps, which prohibit the use
of FNDs for bioapplications by biologists. In this study, we describe
a simple one-pot FND hydrophilization method that comprises coating
of the surface of the nanoparticles with COOH-terminated hyperbranched
polyglycerol (HPG-COOH). HPG-COOH-coated FNDs (FND-HPG-COOHs) were
found to exhibit excellent dispersibility under physiological conditions
despite the thinness of the 5 nm HPG-COOH layer. Biotinylated FND-HPG-COOHs
specifically captured avidin molecules in the absence of nonspecific
protein adsorption. Moreover, we demonstrated that FND-HPG-COOHs conjugated
with antibodies can be used to selectively target integrins in fixed
HeLa cells. In addition, intracellular temperature changes were measured
via optically detected magnetic resonance using FND-HPG-COOHs conjugated
with mitochondrial localization signal peptides. Our one-pot synthetic
method will encourage the broad use of FNDs among molecular and cellular
biologists and pave the way for extensive biological and biomedical
applications of FNDs
Development of Fluorogenic Probes for Quick No-Wash Live-Cell Imaging of Intracellular Proteins
We
developed novel fluorogenic probes for no-wash live-cell imaging
of proteins fused to PYP-tag, which is a small protein tag recently
reported by our group. Through the design of a new PYP-tag ligand,
specific intracellular protein labeling with rapid kinetics and fluorogenic
response was accomplished. The probes crossed the cell membrane, and
cytosolic and nuclear localizations of PYP-tagged proteins without
cell washing were visualized within a 6-min reaction time. The fluorogenic
response was due to the environmental effect of fluorophore upon binding
to PYP-tag. Furthermore, the PYP-tag-based method was applied to the
imaging of methyl-CpG-binding domain localization. This rapid protein-labeling
system combined with the small protein tag and designed fluorogenic
probes offers a powerful method to study the localization, movement,
and function of cellular proteins
Pruning the ALS-Associated Protein SOD1 for in-Cell NMR
To efficiently deliver isotope-labeled
proteins into mammalian cells poses a main challenge for structural
and functional analysis by in-cell NMR. In this study we have employed
cell-penetrating peptides (CPPs) to deliver the ALS-associated protein
superoxide dismutase (SOD1) into HeLa cells. Our results show that,
although full-length SOD1 cannot be efficiently internalized, a variant
in which the active-site loops IV and VII have been truncated (SOD1<sup>ΔIVΔVII</sup>) yields high cytosolic delivery. The reason
for the enhanced delivery of SOD1<sup>ΔIVΔVII</sup> seems
to be the elimination of negatively charged side chains, which alters
the net charge of the CPP-SOD1 complex from neutral to +4. The internalized
SOD1<sup>ΔIVΔVII</sup> protein displays high-resolution
in-cell NMR spectra similar to, but not identical to, those of the
lysate of the cells. Spectral differences are found mainly in the
dynamic β strands 4, 5, and 7, triggered by partial protonation
of the His moieties of the Cu-binding site. Accordingly, SOD1<sup>ΔIVΔVII</sup> doubles here as an internal pH probe, revealing
cytosolic acidification under the experimental treatment. Taken together,
these observations show that CPP delivery, albeit inefficient at first
trials, can be tuned by protein engineering to allow atomic-resolution
NMR studies of specific protein structures that have evaded other
in-cell NMR approaches: in this case, the structurally elusive apoSOD1
barrel implicated as precursor for misfolding in ALS
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High-Sensitivity Rheo-NMR Spectroscopy for Protein Studies
Shear
stress can induce structural deformation of proteins, which
might result in aggregate formation. Rheo-NMR spectroscopy has the
potential to monitor structural changes in proteins under shear stress
at the atomic level; however, existing Rheo-NMR methodologies have
insufficient sensitivity to probe protein structure and dynamics.
Here we present a simple and versatile approach to Rheo-NMR, which
maximizes sensitivity by using a spectrometer equipped with a cryogenic
probe. As a result, the sensitivity of the instrument ranks highest
among the Rheo-NMR spectrometers reported so far. We demonstrate that
the newly developed Rheo-NMR instrument can acquire high-quality relaxation
data for a protein under shear stress and can trace structural changes
in a protein during fibril formation in real time. The described approach
will facilitate rheological studies on protein structural deformation,
thereby aiding a physical understanding of shear-induced amyloid fibril
formation
Cell Cycle-Dependent Turnover of 5-Hydroxymethyl Cytosine in Mouse Embryonic Stem Cells
<div><p>Hydroxymethylcytosine in the genome is reported to be an intermediate of demethylation. In the present study, we demonstrated that maintenance methyltransferase Dnmt1 scarcely catalyzed hemi-hydroxymethylated DNA and that the hemi-hydroxymethylated DNA was not selectively recognized by the SRA domain of Uhrf1, indicating that hydroxymethylcytosine is diluted in a replication-dependent manner. A high level of 5-hydroxymethylcytosine in mouse embryonic stem cells was produced from the methylcytosine supplied mainly by <i>de novo</i>-type DNA methyltransferases Dnmt3a and Dnmt3b. The promoter regions of the <i>HoxA</i> gene cluster showed a high hydroxymethylation level whilst the methylcytosine level was quite low, suggesting that methylated CpG is actively hydroxylated during proliferation. All the results indicate that removal and production of hydroxymethylcytosine are regulated in replication-dependent manners in mouse embryonic stem cells.</p> </div
Substrate/Product-Targeted NMR Monitoring of Pyrimidine Catabolism and Its Inhibition by a Clinical Drug
We report the application of one-dimensional triple-resonance
NMR
to metabolic analysis and thereon-based evaluation of drug activity.
Doubly <sup>13</sup>C/<sup>15</sup>N-labeled uracil ([<sup>15</sup>N1,<sup>13</sup>C6]-uracil) was prepared. Its catabolic (degradative)
conversion to [<sup>13</sup>C3,<sup>15</sup>N4]−β-alanine
and inhibition thereof by gimeracil, a clinical co-drug used with
the antitumor agent 5-fluorouracil, in mouse liver lysates were monitored
specifically using one-dimensional triple-resonance (<sup>1</sup>H–{<sup>13</sup>C–<sup>15</sup>N}) NMR, but not double-resonance (<sup>1</sup>H–{<sup>13</sup>C}) NMR, in a ratiometric manner. The
administration of labeled uracil to a mouse resulted in its non-selective
distribution in various organs, with efficient catabolism to labeled
β-alanine exclusively in the liver. The co-administration of
gimeracil inhibited the catabolic conversion of uracil in the liver.
In marked contrast to <i>in vitro</i> results, however,
gimeracil had practically no effect on the level of uracil in the
liver. The potentiality of triple-resonance NMR in the analysis of <i>in vivo</i> pharmaceutical activity of drugs targeting particular
metabolic reactions is discussed
Dnmt3a and Dnmt3b-dependent 5mC are responsible for the production of 5hmC.
<p>The 5hmC (<b>A</b>) and 5mC (<b>B</b>) contents of J1 (blue bars), <i>Dnmt1</i> (1-KO, red bars), and <i>Dnmt3a</i> and <i>Dnmt3b</i> (3-DKO, light green bars) knockout mESCs were determined by q-PCR in the promoters of five representative 5hmC-enriched genes. The values are the averages + SD determined for three independent genomic DNA samples. </p
Dnmt1, Dnmt3a, Dnmt3b, and Tet1 are recruited to 5hmC-enriched promoters.
<p>The occupancy of Dnmt1, Dnmt3a, and Dnmt3b (<b>A</b>), and Tet1, Tet2, and Tet3 (<b>B</b>) was determined by ChIP-qPCR in the promoters of the 5hmC-enriched genes shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082961#pone-0082961-g004" target="_blank">Figure 4</a>. The values are the averages + SD determined for three independent DNA samples.</p
Enrichment of 5mC and 5hmC in specific promoters.
<p>5hmC (blue bars) and 5mC (red bars) were determined by DNA microarray analysis in the promoters of the <i>Pcdha</i> gene cluster (<b>A</b>), maternal imprinting genes (<b>B</b>), and <i>HoxA</i> gene cluster (<b>C</b>). The abscissas indicate enrichment of 5hmC or 5mC on a log<sub>2</sub> scale.</p
Cell cycle-dependent change in the 5hmC content.
<p><b>A</b>. The 5hmC content in mESCs treated with aphidicolin, hydroxyurea, serum depletion, or nocodazole was determined by β-GT assaying. The values represent the fold change normalized as to that without treatment. The values for each treatment are averages ± SD (n=3). <b>B</b>. The 5hmC content in mESCs sorted by FACS (left panel) was determined (right panel). The values are averages ± SE (n=3). <b>C</b>. The non-synchronized (w/o S) and synchronized mESCs were collected after the indicated times and the 5hmC contents were determined. The left panels show the results of FACS analyses and the right panel the 5hmC content.</p