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1.  Characterization of the Molecular Mechanism of the Bone-Anabolic Activity of Carfilzomib in Multiple Myeloma 
PLoS ONE  2013;8(9):e74191.
Carfilzomib, the next generation of proteasome inhibitor, may increase osteoblast-related markers in patients with multiple myeloma, but the molecular mechanism of its effect on mesenchymal stem cell differentiation to osteoblasts remains unknown. Herein, we demonstrated that carfilzomib significantly promoted mesenchymal stem cell differentiation into osteoblasts. In osteoprogenitor cells and primary mesenchymal stem cells from patients with myeloma, carfilzomib induced increases in alkaline phosphatase activity, matrix mineralization, and calcium deposition via Wnt-independent activation of β-catenin/TCF signaling. Using affinity pull-down assays with immunoblotting analysis and immunofluorescence, we found that carfilzomib induced stabilization of both free and active forms of β-catenin in a time- and dose-dependent manner that was not associated with β-catenin transcriptional regulation. Nuclear translocation of β-catenin protein was associated with TCF transcriptional activity that was independent of the effects of GSK3β-activation and of signaling induced by 19 Wnt ligands, 10 Frizzled receptors, and LRP5/6 co-receptors. Blocking activation of β-catenin/TCF signaling by dominant negative TCF1 or TCF4 attenuated carfilzomib-induced matrix mineralization. Thus, carfilzomib induced osteoblast differentiation via Wnt-independent activation of the β-catenin/TCF pathway. These results provide a novel molecular mechanism critical to understanding the anabolic role of carfilzomib on myeloma-induced bone disease.
PMCID: PMC3774816  PMID: 24066119
2.  Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide 15N–15N and carbonyl 13C–13C dipolar recoupling data 
Recent structural studies of uniformly 15N, 13C-labeled proteins by solid state nuclear magnetic resonance (NMR) rely principally on two sources of structural restraints: (i) restraints on backbone conformation from isotropic 15N and 13C chemical shifts, based on empirical correlations between chemical shifts and backbone torsion angles; (ii) restraints on inter-residue proximities from qualitative measurements of internuclear dipole–dipole couplings, detected as the presence or absence of inter-residue crosspeaks in multidimensional spectra. We show that site-specific dipole–dipole couplings among 15N-labeled backbone amide sites and among 13C-labeled backbone carbonyl sites can be measured quantitatively in uniformly-labeled proteins, using dipolar recoupling techniques that we call 15N-BARE and 13C-BARE (BAckbone REcoupling), and that the resulting data represent a new source of restraints on backbone conformation. 15N-BARE and 13C-BARE data can be incorporated into structural modeling calculations as potential energy surfaces, which are derived from comparisons between experimental 15N and 13C signal decay curves, extracted from crosspeak intensities in series of two-dimensional spectra, with numerical simulations of the 15N-BARE and 13C-BARE measurements. We demonstrate this approach through experiments on microcrystalline, uniformly 15N, 13C-labeled protein GB1. Results for GB1 show that 15N-BARE and 13C-BARE restraints are complementary to restraints from chemical shifts and inter-residue crosspeaks, improving both the precision and the accuracy of calculated structures.
PMCID: PMC3568759  PMID: 22449573
Magic-angle spinning; Protein structure; Pulse sequences
3.  Inflammatory myofibroblastic tumour of the bladder: Case report and review of the literature 
Inflammatory myofibroblastic tumour (IMT) is a rare tumour with malignant potential, and has been described in many major organs. However, bladder location is very uncommon. We report the case of a 23-year-old woman who presented with painless gross hematuria for 2 weeks. Contrast-enhanced computed tomography revealed a bladder tumour. The patient underwent an open partial cystectomy and the final pathologic diagnosis was IMT of bladder. Typical IMTs can be locally aggressive, therefore close follow-up is necessary.
PMCID: PMC3650765  PMID: 23671533
4.  Signal enhancement for the sensitivity-limited solid state NMR experiments using a continuous, non-uniform acquisition scheme 
We describe a sampling scheme for the two-dimensional (2D) solid state NMR experiments, which can be readily applied to the sensitivity-limited samples. The sampling scheme utilizes continuous, non-uniform sampling profile for the indirect dimension, i.e. the acquisition number decreases as a function of the evolution time (t1) in the indirect dimension. For a beta amyloid (Aβ) fibril sample, we observed overall 40 ~ 50% signal enhancement by measuring the cross peak volume, while the cross peak linewidths remained comparable to the linewidths obtained by regular sampling and processing strategies. Both the linear and Gaussian decay functions for the acquisition numbers result in similar percentage of increment in signal. In addition, we demonstrated that this sampling approach can be applied with different dipolar recoupling approaches such as radiofrequency assisted diffusion (RAD) and finite-pulse radio-frequency-driven recoupling (fpRFDR). This sampling scheme is especially suitable for the sensitivity-limited samples which require long signal averaging for each t1 point, for instance the biological membrane proteins where only a small fraction of the sample is isotopically labeled.
PMCID: PMC3206163  PMID: 21930405
solid state NMR; non-uniform sampling; sensitivity-limited sample; diluted sample
5.  Early immune outcome of retroperitoneal laparoscopic radical nephrectomy for localized renal cell carcinoma: a prospective, randomized study 
We evaluated differences in cytokine responses and T-lymphocyte subsets following retroperitoneal laparoscopic and conventional open radical nephrectomies for localized renal cell carcinoma (RCC).
A total of 62 patients with T1N0M0 staged RCC were randomized to either retro-laparoscopic (n = 31) or open (n = 31) radical nephrectomy. Plasma levels of interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-alpha (TNF-α) were measured separately by enzyme linked immunosorbent assay (ELISA) preoperatively and on postoperative days 1 and 5. Levels of CD3+, CD4+ and CD8+ as well as the CD4+:CD8+ ratio were acquired by flow cytometry at the same time points.
Levels of IL-1β, IL-6 and TNF-α increased significantly compared to preoperative values in both groups (p < 0.05) on postoperative day 1, and all the parameters in the open group were significantly higher than those in the retro-laparoscopy group (p < 0.05). On postoperative day 1, the levels of CD3+ and CD4+ and the CD4+:CD8+ ratio decreased markedly compared to preoperative values for both groups (p < 0.05). Elevations of the CD4+:CD8+ ratio in the retro-laparoscopy group (p < 0.05) and the CD8+ level in the open group (p < 0.05) were observed when compared with the other group. On postoperative day 5, the levels of CD3+ and CD4+ and the CD4+:CD8+ ratio in the retro-laparoscopy group, as well as the level of CD8+ in the open group, returned to about preoperative levels (p < 0.05). Follow-up ranged from 4 to 14 months postoperatively in all 62 patients with a 100% cancer-specific survival rate in both groups.
Retroperitoneal laparoscopic radical nephrectomy is associated with the milder cytokine responses caused by trauma and inflammation and the better preserved distribution of T-lymphocytes.
PMCID: PMC3529729  PMID: 22060731
6.  Detection of Two Zoonotic Babesia microti Lineages, the Hobetsu and U.S. Lineages, in Two Sympatric Tick Species, Ixodes ovatus and Ixodes persulcatus, Respectively, in Japan 
The species Babesia microti, commonly found in rodents, demonstrates a high degree of genetic diversity. Three lineages, U.S., Kobe, and Hobetsu, are known to have zoonotic potential, but their tick vector(s) in Japan remains to be elucidated. We conducted a field investigation at Nemuro on Hokkaido Island and at Sumoto on Awaji Island, where up to two of the three lineages occur with similar frequencies in reservoirs. By flagging vegetation at these spots and surrounding areas, 4,010 ticks, comprising six species, were collected. A nested PCR that detects the 18S rRNA gene of Babesia species revealed that Ixodes ovatus and I. persulcatus alone were positive. Lineage-specific PCR for rRNA-positive samples demonstrated that I. ovatus and I. persulcatus carried, respectively, the Hobetsu and U.S. parasites. No Kobe-specific DNA was detected. Infected I. ovatus ticks were found at multiple sites, including Nemuro and Sumoto, with minimum infection rates (MIR) of ∼12.3%. However, all I. persulcatus ticks collected within the same regions, a total of 535, were negative for the Hobetsu lineage, indicating that I. ovatus, but not I. persulcatus, was the vector for the lineage. At Nemuro, U.S. lineage was detected in 2 of 139 adult I. persulcatus ticks (MIR, 1.4%), for the first time, while 48 of I. ovatus ticks were negative for that lineage. Laboratory experiments confirmed the transmission of Hobetsu and U.S. parasites to hamsters via I. ovatus and I. persulcatus, respectively. Differences in vector capacity shown by MIRs at Nemuro, where the two species were equally likely to acquire either lineage of parasite, may explain the difference in distribution of Hobetsu throughout Japan and U.S. taxa in Nemuro. These findings are of importance in the assessment of the regional risk for babesiosis in humans.
PMCID: PMC3346458  PMID: 22389378
7.  A general Monte Carlo/simulated annealing algorithm for resonance assignment in NMR of uniformly labeled biopolymers 
Journal of biomolecular NMR  2011;50(3):267-276.
We describe a general computational approach to site-specific resonance assignments in multidimensional NMR studies of uniformly 15N,13C-labeled biopolymers, based on a simple Monte Carlo/simulated annealing (MCSA) algorithm contained in the program MCASSIGN2. Input to MCASSIGN2 includes lists of multidimensional signals in the NMR spectra with their possible residue-type assignments (which need not be unique), the biopolymer sequence, and a table that describes the connections that relate one signal list to another. As output, MCASSIGN2 produces a high-scoring sequential assignment of the multidimensional signals, using a score function that rewards good connections (i.e., agreement between relevant sets of chemical shifts in different signal lists) and penalizes bad connections, unassigned signals, and assignment gaps. Examination of a set of high-scoring assignments from a large number of independent runs allows one to determine whether a unique assignment exists for the entire sequence or parts thereof. We demonstrate the MCSA algorithm using two-dimensional (2D) and three-dimensional (3D) solid state NMR spectra of several model protein samples (α-spectrin SH3 domain and protein G/B1 microcrystals, HET-s218–289 fibrils), obtained with magic-angle spinning and standard polarization transfer techniques. The MCSA algorithm and MCASSIGN2 program can accommodate arbitrary combinations of NMR spectra with arbitrary dimensionality, and can therefore be applied in many areas of solid state and solution NMR.
PMCID: PMC3199575  PMID: 21710190
Sequential assignment; Solid state NMR; Magic-angle spinning; Multidimensional spectroscopy
8.  Structural evolution of Iowa-mutant β-amyloid fibrils from polymorphic to homogeneous states under repeated seeded growth 
Structural variations in β-amyloid fibrils are potentially important to the toxicity of these fibrils in Alzheimer's disease (AD). We describe a repeated seeding protocol that selects a homogeneous fibril structure from a polymorphic initial state in the case of 40-residue β-amyloid fibrils with the Asp23-to-Asn, or Iowa, mutation (D23N-Aβ1-40). We use thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and solid state nuclear magnetic resonance (NMR) to track the evolution of fibril structure through multiple generations under this protocol. The data show that: (i) repeated seeding selectively amplifies a single D23NAβ1-40 fibril structure that can be a minor component of the initial polymorphic state; (ii) the final structure is highly sensitive to growth conditions, including pH, temperature, and agitation; (iii) although the initial state can include fibrils that contain both antiparallel and parallel β-sheets, the final structures contain only parallel β-sheets, suggesting that antiparallel β-sheet structures are thermodynamically and kinetically metastable. Additionally, our data demonstrate that ThT fluorescence enhancements, which are commonly used to monitor amyloid fibril formation, vary strongly with structural variations, even among fibrils comprised of the same polypeptide. Finally, we present a simple mathematical model that describes the structural evolution of fibril samples under repeated seeding.
PMCID: PMC3060308  PMID: 21355554
Alzheimer's disease; solid state NMR; electron microscopy; thioflavin T fluorescence; amyloid growth mechanism; amyloid growth kinetics
9.  HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics 
Journal of molecular biology  2010;402(1):139-153.
This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus KC, a measure of membrane stiffness, and wide-angle x-ray scattering yielded the Sxray orientational order parameter. Both FP23 and FPtri decreased KC and Sxray considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion.
PMCID: PMC2940274  PMID: 20655315
fusion peptide; trimer; T-cell mimic; structure; bending modulus
10.  Association of insulin-like growth factor-1 with thyroid nodules 
Oncology Letters  2011;2(6):1297-1301.
The aim of the present study was to investigate the relationship between insulin-like growth factor-1 (IGF-1) and thyroid nodules. A total of 56 patients with thyroid nodules confirmed by physical examination and ultrasound screening were randomly selected. The patients were divided into three groups by radionuclide scan: the hot nodule group (group 1, n=18); the cold and solid nodule group (group 2, n=18); and the cold and cystic nodule group (group 3, n=20). Cystic fluid samples from patients with cystic cold thyroid nodules were defined as group 4. A control group of 18 healthy adults matched for age, gender and body mass index (group 0) was also included. For all participants, levels of the thyroid hormones, TT3, TT4, TSH and IGF-1, were determined by radioimmunoassay. The measurement data were expressed as the mean ± standard deviation (SD). The analysis of variance was performed by the t-test and the correlation analysis was performed by linear regression. The serum levels of IGF-1 in the solid cold nodule group were significantly higher than those in the hot nodule group (P<0.05). Serum levels of IGF-1 in the cystic cold nodule group were significantly lower than those in the control group (P<0.05). The serum IGF-1 levels in the cystic fluid were significantly lower than those in the cystic cold nodule (P<0.05) and the control groups (P<0.05). Additionally, the mean serum IGF-1 level in patients with thyroid adenoma was significantly higher than that in the control group (P<0.05). The serum IGF-1 level may not be involved in the pathogenesis of hot thyroid nodules and cold and cystic thyroid nodules; however, it may play a significant role in the pathogenesis of certain solid cold thyroid nodules.
PMCID: PMC3406504  PMID: 22848305
thyroid nodule; IGF-1; pathogenesis
11.  Nuclear Magnetic Resonance Evidence for Retention of a Lamellar Membrane Phase with Curvature in the Presence of Large Quantities of the HIV Fusion Peptide 
Biochimica et biophysica acta  2009;1798(2):194-201.
The HIV fusion peptide (HFP) is a biologically relevant model system to understand virus/host cell fusion. 2H and 31P NMR spectroscopy were applied to probe the structure and motion of membranes with bound HFP and with a lipid headgroup and cholesterol composition comparable to that of membranes of host cells of HIV. The lamellar phase was retained for a variety of highly fusogenic HFP constructs as well as a non-fusogenic HFP construct and for the influenza virus fusion peptide. The lamellar phase is therefore a reasonable structure for modeling the location of HFP in lipid/cholesterol dispersions. Relative to no HFP, membrane dispersions with HFP had faster 31P transverse relaxation and faster transverse relaxation of acyl chain 2H nuclei closest to the lipid headgroups. Relative to no HFP, mechanically aligned membrane samples with HFP had broader 31P signals with a larger fraction of unoriented membrane. The relaxation and aligned sample data are consistent with bilayer curvature induced by the HFP which may be related to its fusion catalytic function. In some contrast to the subtle effects of HFP on a host-cell-like membrane composition, an isotropic phase was observed in dispersions rich in phosphatidylethanolamine lipids and with bound HFP.
PMCID: PMC2812645  PMID: 19616505
HIV; fusion; peptide; NMR; curvature; membrane
12.  The HIV Fusion Peptide and its Cross-Linked Oligomers: Efficient Syntheses, Significance of the Trimer in Fusion Activity, Correlation of β Strand Conformation with Membrane Cholesterol, and Proximity to Lipid Headgroups† 
Biochemistry  2009;48(2):289-301.
For enveloped viruses such as HIV, a ~20-residue N-terminal fusion peptide domain in the envelope protein binds to target cell membranes and plays a key role in fusion between the viral and cellular membranes during infection. The chemically synthesized HIV fusion peptide or “HFP” catalyzes fusion between membrane vesicles and is a useful model system to understand some aspects of HIV fusion. Previous studies have shown a common trimeric state for envelope protein from several different viruses including HIV and in this study, practical high-yield syntheses are reported for HFP monomer (HFPmn) and chemically cross-linked HFP dimer (HFPdm), trimer (HFPtr), and tetramer (HFPte). The vesicle fusion rates per strand were ordered HFPmn < HFPdm < HFPtr ≈ HFPte and suggested that HFPtr is the smallest catalytically efficient oligomer. Solid-state NMR measurements of 13CO chemical shifts were carried out in constructs labeled at either Ala-6 or Ala-15. For all constructs associated with cholesterol-containing membranes, the chemical shifts of both residues correlated with β strand conformation while association with membranes without cholesterol resulted in a mixture of helical and β strand conformations. The dependence of fusion rate on oligomer size is independent of membrane cholesterol content, so one interpretation of the data is fusion activity of both helical and β strand conformations. Membrane location may be a determinant of fusion activity and for all constructs in both conformations, a large fraction of the Ala-15 13COs were 5–6 Å from the 31Ps in the lipid headgroups, while the Ala-6 13COs were more distant.
PMCID: PMC2680607  PMID: 19093835
fusion peptide; HIV; NMR; membrane location; oligomer; cholesterol; synthesis
13.  Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide to Lipid Distances Reveal Intimate Contact of β Strand Peptide with Membranes and Close Proximity of the Ala-14 to Gly-16 Region with Lipid Headgroups 
Biochemistry  2007;46(17):4997-5008.
Human immunodeficiency virus (HIV) infection begins with fusion between viral and host cell membranes and is catalyzed by the HIV gp41 fusion protein. The ~20 N-terminal apolar residues of gp41 are called the HIV fusion peptide (HFP), interact with the host cell membrane, and play a key role in fusion. In this study, the membrane location of peptides which contained the HFP sequence AVGIGALFLGFLGAAGSTMGARS was probed in samples containing either only phospholipids or phospholipids and cholesterol. Four HFPs were examined which each contained 13CO labeling at three sequential residues between G5 and G16. The 13CO chemical shifts indicated that HFP had predominant β strand conformation over the labeled residues in the samples. The internuclear distances between the HFP 13COs and the lipid 31Ps were measured using solid-state nuclear magnetic resonance rotational-echo double resonance experiments. The closest 13CO-31P distances of 5–6 Å were observed for HFP labeled between A14 and G16 and correlated with intimate association of β strand HFP and membranes. These results were confirmed with measurements using HFPs singly 13CO labeled at A6 or A14. To our knowledge, these data are the first measurements of distances between HIV fusion peptide nuclei and lipid P and qualitative models of membrane location of oligomeric β strand HFP are presented which are consistent with the experimental data. Observation of intimate contact between β strand HFP and membranes provides rationale for further investigation of the relationship between structure and fusion activity for this conformation.
PMCID: PMC2631438  PMID: 17417873
HIV; fusion peptide; membrane; carbonyl; phosphorus; REDOR; NMR

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