Development of a novel multiplex digital PCR-based method for the detection of HTLV-1 proviral deletion.

The human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, integrates into host DNA and causes adult T-cell leukemia/lymphoma (ATL) in some individuals. Two types of defective proviruses, Type 1 and Type 2, are often observed in ATL cells. Here, we developed a 3-plex digital PCR (dPCR) method to detect HTLV-1 proviral deletions by comparing the ratios of copy numbers quantified using specific primer-probes for the LTR, pol, and pX regions.

Calcium-dependent reversible coaggregation activity of C-reactive protein and M-ficolin.

C-reactive protein (CRP) and M-ficolin are the pattern recognition proteins of the innate immune system. In this report, a mixture of CRP and M-ficolin reversibly co-aggregated in a calcium-dependent manner. This coaggregation was enhanced at low pH (6.

RAISING is a high-performance method for identifying random transgene integration sites.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites.

Validation of HPLC Method for Determination of Histamine in Human Immunoglobulin Formulations.

Background: Histamine fixed-immunoglobulin formulations, which consisted of 0.15 µg of histamine dihydrochloride and 12 mg of human immunoglobulin in a vial, are used for anti-allergic treatments, and controlling the amounts of histamine in the formulations is essential to avoid histamine intoxication.

Objective: A high-performance liquid chromatography (HPLC) method for determination of histamine contents of the formulations was established and validated.

A high-throughput detection method for the clonality of Human T-cell leukemia virus type-1-infected cells in vivo.

Approximately 10-20 million of Human T-cell leukemia virus type-1 (HTLV-1)-infected carriers have been previously reported, and approximately 5% of these carriers develop adult T-cell leukemia/lymphoma (ATL) with a characteristic poor prognosis. In Japan, Southern blotting has long been routinely performed for detection of clonally expanded ATL cells in vivo, and as a confirmatory diagnostic test for ATL. However, alternative methods to Southern blotting, such as sensitive, quantitative, and rapid analytical methods, are currently required in clinical practice.

¹H, ¹³C and ¹⁵N backbone resonance assignments of the monomeric human M-ficolin fibrinogen-like domain secreted by Brevibacillus choshinensis.

M-ficolin, which forms trimer-based multimers, is a pathogen-recognition protein in the innate immune system, and it binds to ligands through its fibrinogen-like (FBG) domain. As the first step toward the elucidation of the molecular basis for pathogen-recognition by the M-ficolin multimers, we assigned the backbone resonances of the monomeric mutant of the M-ficolin FBG domain, recombinantly expressed by Brevibacillus choshinensis. Like the wild-type trimeric FBG domain, the monomeric FBG domain also requires His251, His284 and His297 for the ligand-binding activity, as judged by mutational analyses using zonal affinity chromatography.

Intramolecular allosteric interaction in the phospholipase C-δ1 pleckstrin homology domain.

Protein activities are generally regulated by intramolecular allosteric interactions, by which spatially separated sites in a protein molecule communicate. Intramolecular allosteric interactions in the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain were investigated by solution NMR spectroscopy for selectively [α-(15)N]Lys-labeled proteins. The results of NMR analyses indicated that the binding of inositol 1,4,5-trisphosphate (IP3) to the protein induces local environmental changes at all lysine residues, including residues such as Lys-43 spatially separated from the specific IP3 binding site consisting of Lys-30, Lys-32, and Lys-57.

Analysis of the phospholipase C-δ1 pleckstrin homology domain using native polyacrylamide gel electrophoresis.

The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP(3)) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP(3) binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP(3) and in thermal destabilization of the IP(3)-binding state.

A structure-based mechanism for benzalacetone synthase from Rheum palmatum.

Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 A resolution.

Binding site of C-reactive protein on M-ficolin.

The binding abilities of human C-reactive protein (CRP) with the C-terminal fibrinogen-like (FBG) domain and the full-length form of human M-ficolin were investigated by pull-down and zonal affinity chromatography analyses. Pull-down assays using an N-acetyl-D-glucosamine (GlcNAc)-agarose column demonstrated that CRP binds to the trimeric FBG domains, and that the GlcNAc-binding ability of the FBG domain is unaffected by CRP binding. Interestingly, the full-length M-ficolin, comprising the N-terminal collagen-like (COL) and C-terminal FBG domains, displayed lower affinity for CRP, and the monomeric FBG domain showed virtually no binding to CRP, as qualitatively judged by zonal affinity chromatography using a GlcNAc column.

Suppressed or recovered intensities analysis in site-directed (13)C NMR: assessment of low-frequency fluctuations in bacteriorhodopsin and D85N mutants revisited.

The first proton transfer of bacteriorhodopsin (bR) occurs from the protonated Schiff base to the anionic Asp 85 at the central part of the protein in the L to M states. Low-frequency dynamics accompanied by this process can be revealed by suppressed or recovered intensities (SRI) analysis of site-directed (13)C solid-state NMR spectra of 2D crystalline preparations. First of all, we examined a relationship of fluctuation frequencies available from [1-(13)C]Val- and [3-(13)C]Ala-labeled preparations, by taking the effective correlation time of internal methyl rotations into account.

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis.

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the 15N-AATS-labeled protein, the transformed B. choshinensis was cultured in 15N-labeled amino acid-containing C.

Histidine-regulated activity of M-ficolin.

Human M-ficolin is a pathogen-associated molecular recognition molecule in the innate immune system, and it binds to some sugars, such as GlcNAc (N-acetylglucosamine), on pathogen surfaces. From previous structural and functional studies of the FD1 (M-ficolin fibrinogen-like domain), we proposed that the ligand-binding region of FD1 exists in a conformational equilibrium between active and non-active states depending on three groups with a pK(a) of 6.2, which are probably histidine residues, and suggested that the 2-state conformational equilibrium as well as the trimer formation contributes to the discrimination mechanism between self and non-self of FD1 [Tanio, M.

Trimeric structure and conformational equilibrium of M-ficolin fibrinogen-like domain.

Ficolins are pathogen-recognition molecules in innate immune systems. The crystal structure of the human M-ficolin recognition domain (FD1) has been determined at 1.9 A resolution, and compared with that of the human fibrinogen gamma fragment, tachylectin-5A, L-ficolin and H-ficolin.

Crystallization and preliminary crystallographic analysis of a plant type III polyketide synthase that produces benzalacetone.

Benzalacetone synthase (BAS) from Rheum palmatum is a plant-specific type III polyketide synthase that catalyzes the one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide 4-(4-hydroxyphenyl)-but-3-en-2-one. Recombinant BAS expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belong to space group P2(1), with unit-cell parameters a = 54.

Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein.

Tau-tubulin kinase 2 (TTBK2) is a Ser/Thr kinase that putatively phosphorylates residues Ser208 and Ser210 (numbered according to a 441-residue human tau isoform) in tau protein. Functional analyses revealed that a recombinant kinase domain (residues 1-331) of human TTBK2 expressed in insect cells with a baculovirus overexpression system retains kinase activity for tau protein. The kinase domain of TTBK2 was crystallized using the hanging-drop vapour-diffusion method.

Surface and dynamic structures of bacteriorhodopsin in a 2D crystal, a distorted or disrupted lattice, as revealed by site-directed solid-state 13C NMR.

The 3D structure of bacteriorhodopsin (bR) obtained by X-ray diffraction or cryo-electron microscope studies is not always sufficient for a picture at ambient temperature where dynamic behavior is exhibited. For this reason, a site-directed solid-state 13C NMR study of fully hydrated bR from purple membrane (PM), or a distorted or disrupted lattice, is very valuable in order to gain insight into the dynamic picture. This includes the surface structure, at the physiologically important ambient temperature.

Trivalent recognition unit of innate immunity system: crystal structure of trimeric human M-ficolin fibrinogen-like domain.

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9A resolution.

Overexpression, purification and preliminary crystallographic analysis of human M-ficolin fibrinogen-like domain.

Ficolins, which are comprised of a collagen-like domain and a fibrinogen-like domain, are a kind of pattern-recognition molecule for pathogens in the innate immunity system. To investigate the molecular mechanism of the discrimination between self and non-self by ficolins, human M-ficolin fibrinogen-like domain (FD1), which contains the ligand-binding site, was overexpressed in Pichia pastoris, purified and crystallized using the vapour-diffusion method at 293 K. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 55.

Direct evidence of interaction of a green tea polyphenol, epigallocatechin gallate, with lipid bilayers by solid-state Nuclear Magnetic Resonance.

The interaction of a tea catechin, epigallocatechin gallate (EGCg), with the model membrane of dimyristoylphosphatidylcholine (DMPC) was studied by solid-state (31)P and (2)H NMR. The (31)P chemical shift anisotropy of the DMPC phosphate group decreased on addition of EGCg. The (2)H NMR spectrum of [4-(2)H]EGCg, which is deuterated at the 4-position, in the DMPC liposomes gave deuterium nuclei with much smaller quadrupole splittings than those in the solid phase.

Significance of low-frequency local fluctuation motions in the transmembrane B and C alpha-helices of bacteriorhodopsin, to facilitate efficient proton uptake from the cytoplasmic surface, as revealed by site-directed solid-state 13C NMR.

13C NMR spectra of [1-13C]Val- or -Pro-labeled bacteriorhodopsin (bR) and its single or double mutants, including D85N, were recorded at various pH values to reveal conformation and dynamics changes in the transmembrane alpha-helices, in relation to proton release and uptake between bR and the M-like state caused by modified charged states at Asp85 and the Schiff base (SB). It was found that the D85N mutant acquired local fluctuation motion with a frequency of 10(4) Hz in the transmembrane B alpha-helix, concomitant with deprotonation of SB in the M-like state at pH 10, as manifested from a suppressed 13C NMR signal of the [1-13C]-labeled Val49 residue. Nevertheless, local dynamics at Pro50 neighboring with Val49 turned out to be unchanged, irrespective of the charged state of SB as viewed from the 13C NMR of [1-13C]-labeled Pro50.

Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with [1-13C]amino acid residues.

We have so far demonstrated that well-resolved and site-specifically assigned (13)C peaks as recorded by site-directed NMR study on (13)C-labeled membrane proteins can serve as a convenient probe to reveal their local conformation and dynamics. We attempted here to clarify the extent to which (13)C NMR spectra of (13)C-labeled fully hydrated bacteriorhodopsin (bR) as a typical membrane protein are visible or well resolved in the presence of inherent fluctuation motions with frequency of 10(2)-10(8) Hz, especially at the membrane surfaces. Accordingly, we estimated the relative proportion of (13)C NMR signals from the surface areas with and without peak suppression by the accelerated transverse relaxation effect by surface-bound Mn(2+) ions, which could be effective for residues within 8.