Probing Short-Range Interactions in Isostructural Hydrate and Perhydrate of Dabrafenib by Magic-Angle Spinning Solid-State NMR.

Dabrafenib (DBF), an anticancer drug, exhibits isostructural properties in its hydrate (DBF⊃H2O) and perhydrate (DBF⊃H2O2) forms, as revealed by single-crystal X-ray diffraction. Despite the H2O and H2O2 solvent molecules occupying identical locations, the two polymorphs have different thermal behaviors. In general, determination of stoichiometry of H2O in the perhydrate crystals is difficult due to the presence of both H2O and H2O2 in the same crystal voids. This study utilizes magic-angle spinning (MAS) solid-state NMR (SSNMR) combined with gauge-included projector augmented wave calculations to characterize the influence of solvent molecules on the local environment in pseudopolymorphs. SSNMR experiments were employed to assign 1H, 13C, and 15N peaks and identify spectral differences in the isostructural pseudopolymorphs. Proton spectroscopy at fast MAS was used to identify and quantify H2O2/H2O in DBF⊃H2O2 (mixed hydrate/perhydrate). 1H-1H dipolar-coupling-based experiments were recruited to confirm the 3D molecular packing of solvent molecules in DBF⊃H2O and DBF⊃H2O2. Homonuclear (1H-1H) and heteronuclear (1H-14N) distance measurements, in conjunction with diffraction structures and optimized hydrogen atom positions by density functional theory, helped decipher local interactions of H2O2 with DBF and their geometry in DBF⊃H2O2. This integrated X-ray structure, quantum chemical calculations, and NMR study of pseudopolymorphs offer a practical approach to scrutinizing crystallized solvent interactions in the crystal lattice even without high-resolution crystal structures or artificial sample enrichment.

Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One.

In practice and despite Anfinsen's dogma, the refolding of recombinant multiple SS-bonded proteins is famously difficult because misfolded species with non-native SS-bonds appear upon the oxidization of their cysteine residues. On the other hand, single SS-bond proteins are thought to be simple to refold because their cysteines have only one SS-bond partner. Here, we report that dengue 4 envelope protein domain 3 (DEN4 ED3), a single SS-bonded protein can be irreversibly trapped into a misfolded species through the formation of its sole intramolecular SS-bond. The misfolded species had a much lower solubility than the native one at pHs higher than about 7, and circular dichroism measurements clearly indicated that its secondary structure content was different from the native species. Furthermore, the peaks in the Heteronuclear Single Quantum Correlation spectroscopy (HSQC) spectrum of DEN4 ED3 from the supernatant fraction were sharp and well dispersed, reflecting the beta-sheeted native structure, whereas the spectrum of the precipitated fraction showed broad signals clustered near its center suggesting no or little structure and a strong tendency to aggregate. The two species had distinct biophysical properties and could interconvert into each other only by cleaving and reforming the SS-bond, strongly suggesting that they are topologically different. This phenomenon can potentially happen with any single SS-bonded protein, and our observation emphasizes the need for assessing the conformation and biophysical properties of bacterially produced therapeutic proteins in addition to their chemical purities.

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation.

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.

Aminophylline, administered at usual doses for rodents in pharmacological studies, induces hippocampal neuronal cell injury under low tidal volume hypoxic conditions in guinea-pigs.

OBJECTIVES: To establish whether aminophylline, administered at usual doses for rodents in pharmacological studies, induces brain injury in systemic hypoxaemia in guinea-pigs. METHODS: A hypoxaemia (partial oxygen tension of arterial blood (PaO2) = 40-60 mmHg) model was developed by low tidal volume mechanical ventilation in guinea-pigs. KEY FINDINGS: Under hypoxic conditions, aminophylline significantly increased the concentration of brain-specific creatine kinase in the serum in a dose- and time-dependent manner. A reduced number of hippocampal neuronal cells in the CA1 region, an increase in the concentration of neuron-specific enolase (NSE) in cerebrospinal fluid (CSF), an increase in lipid hydroperoxides and a decrease in the ratio of glutathione to glutathione disulfide in the brain tissues were also observed. These effects were not observed when aminophylline at the same doses was administered under normoxic conditions (PaO2 = 80-100 mmHg). There was no difference in either serum or CSF concentrations of theophylline between normoxic and hypoxic conditions. Another methylxanthine, caffeine, did not increase the concentration of NSE in CSF. CONCLUSIONS: Aminophylline potentially induces brain damage under hypoxic conditions. We suggest that aminophylline treatment has adverse effects in patients with hypoxaemia subsequent to respiratory disorders such as asthma.

Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus.

Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q(n), n = 5-10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q(n) peptide that stained positive to ThT was Q(6). Two types of ThT-binding sites, a high-affinity site (k(d1) = 0.1-0.17 µM) and a low-affinity site (k(d2) = 5.7-7.4 µM), were observed in short polyQs (n = 6-9). (13)C{(2)H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q(8) peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d(3)]ThT and natural abundance Q(8) or residue-specific [1,2-(13)C(2)] labeled Q(8)s. (13)C{(2)H}REDOR difference spectra of the [3-Me-d(3)]ThT/natural abundance Q(8) (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. (13)C{(2)H}DQF-REDOR experiments of [3-Me-d(3)]ThT/residue-specific [1,2-(13)C(2)] labeled Q(8) (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions.

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell 'stemness' and tumorigenesis. Msi1 reportedly binds to the 3'-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between ß1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins.

Leg ulcer caused by Mycobacterium ulcerans ssp. shinshuense infection.

BACKGROUND: An 81-year-old man presented with a skin ulcer on the left forearm caused by infection with Mycobacterium ulcerans ssp. shinshuense. The patient first noticed the subcutaneous nodule with an undermined ulcer and areola on the left forearm without any episode of trauma. METHODS: The rod-shaped and acid-fast bacteria taken from the ulcerative lesion were positive for Ziehl-Neelsen staining. The bacterial colony was cultured on Ogawa slant egg medium at 28 degrees C. RESULTS: A clinical diagnosis of Mycobacterium infection was made. For therapy, in addition to oral clarithromycin, topical sulfadiazine silver and hyperthermia were used. One month after starting treatment, topical treatment was changed to U-pasta (sucrose, povidone-iodine). Four months after the onset of the disease, bacterial colonies composed of scotochromogens were identified as Mycobacterium marinum by the DNA-DNA hybridization (DDH) method. The growth speed and characteristics of the bacterial colonies were different from those of Mycobacterium marinum. CONCLUSIONS: This pathogenetic bacterium was finally identified as Mycobacterium ulcerans ssp. shinshuense by the polymerase chain reaction method and 16S rRNA gene sequencing. Nine months after the onset of the disease, the ulcer was re-epithelialized with a residual scar.

Dynamic inter-subunit interactions in thermophilic F(1)-ATPase subcomplexes studied by cross-correlated relaxation-enhanced polarization transfer NMR.

F(1)-ATPase is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this property is the alpha(3)beta(3)gamma complex (351 kDa). For investigation of such a huge system by means of solution NMR, we have explored a suitable NMR method using F(1)-ATPase subcomplexes from a thermophilic Bacillus PS3 including an alpha(3)beta(3) hexamer (319 kDa). Pulse sequences for large molecules, effects of deuteration and simplification of the spectra were examined in this work. Since the beta subunit includes the catalytic site, this was the target of the analysis in this work. The combination of [(15)N,(1)H]-CRINEPT-HMQC-[(1)H]-TROSY, deuteration of both alpha and beta subunits, and segmental isotope-labeling was found essential to analyze such a huge and complex molecular system. Utilizing this method, subcomplexes composed of alpha and beta subunits were investigated in terms of inter-subunit interactions. It turned out that there is equilibrium among monomers, heterodimers and the alpha(3)beta(3) hexamers in solution. The rate of exchange between the dimer and hexamer is in the slow regime on the NMR time scale. In chemical shift perturbation experiments, the N-terminal domain was found to be involved in strong inter-subunit interactions. In contrast, the C-terminal domain was found to be mobile even in the hexamer.

Evaluation of serum fetuin-A relationships with biochemical parameters in patients on hemodialysis.

BACKGROUND: Complications associated with atherosclerosis in dialysis patients are attracting attention. Fetuin-A, a circulating calcium-regulatory glycoprotein that inhibits vascular calcification, is associated with inflammation and outcome in dialysis patients. In this study, the relation between serum fetuin-A concentration and biochemical parameters in patients on hemodialysis was investigated. METHODS: Forty hemodialysis patients, 22 men and 18 women, aged 57 +/- 12 years; and 20 controls, 10 men and 10 women, aged 50 +/- 10 years, participated in this study. We measured serum fetuin-A by enzyme-linked immunosorbent assay. The biochemical parameters of serum albumin, alkaline phosphatase, calcium, phosphate, intact parathyroid hormone, total cholesterol, triglyceride, lipoprotein (a), brain natriuretic peptide (BNP), highly sensitive C-reactive protein (hsCRP), hemoglobin, and hematocrit in whole blood were also measured before starting dialysis sessions. Other parameters included the cardio ankle vascular index, age, mean arterial pressure, total weekly urea clearance (Kt/V), smoking habit, body mass index (BMI), and duration of dialysis. These variables were included in simple regression analysis. RESULTS: Levels of serum fetuin-A in the hemodialysis patients (331 +/- 55 microg/ml) were significantly lower than those in the healthy controls (361 +/- 55 microg/ml; P < 0.05). There was a negative correlation between serum fetuin-A levels and duration of dialysis (r = -0.37, P < 0.01), BNP (r = -0.37, P < 0.001), and hsCRP (r = -0.40, P < 0.01), and a positive association with serum albumin (r = 0.31, P < 0.05). CONCLUSIONS: These data suggest that a low fetuin-A level is a useful predictor of malnutrition and inflammation, as well as being a useful predictor of the cardiac failure caused by an increased ventricular load in hemodialysis patients.

Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.

OBJECTIVES: Macrophages (Mphis) have various functions and play a critical role in host defense and the maintenance of homeostasis. Mphis exist in every tissue in the body, but Mphis from different tissues exhibit a wide range of phenotypes with regard to their morphology, cell surface antigen expression and function, and are called by different names. However, the precise mechanism of the generation of macrophage heterogeneity is not known. In the present study, the authors examined the functional heterogeneity of Mphis generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF). METHODOLOGY: CD14 positive human monocytes (Mos) were incubated with M-CSF and GM-CSF for 6-7 days to stimulate the generation of M-CSF-induced monocyte-derived Mphis (M-Mphis) and GM-CSF-induced monocyte-derived Mphis (GM-Mphis), respectively. The expression of cell surface antigens and several functions such as antigen presenting cell activity, susceptibility to oxidant stress, and the susceptibility to HIV-1 and mycobacterium tuberculosis infection were examined. RESULTS: GM-Mphis and M-Mphis are distinct in their morphology, cell surface antigen expression, and functions examined. The phenotype of GM-Mphis closely resembles that of human Alveolar-Mphis (A-Mphis), indicating that CSF-induced human monocyte-derived Mphis are useful to clarify the molecular mechanism of heterogeneity of human Mphis, and GM-Mphis will become a model of human A-Mphis.

Identification of the N- and C-terminal substrate binding segments of ferredoxin-NADP+ reductase by NMR.

Ferredoxin-NADP(+) reductase (FNR) catalyzes the reduction of NADP(+) through the formation of an electron transfer complex with ferredoxin. To gain insight into the interaction of this enzyme with substrates at both ends of the polypeptide chain, we performed NMR analyses of a 314-residue maize leaf FNR with a nearly complete assignment of the backbone resonances. The chemical shift perturbation upon formation of the complex indicated that a flexible N-terminal region of FNR contributed to the interaction with maize ferredoxin, and an analysis of N-terminally truncated mutants of FNR confirmed the importance of this region for the binding of ferredoxin. Comparison between the spectra of FNR in the NADP(+)- and inhibitor-bound states also revealed that the nicotinamide moiety of NADP(+) was accessible to the C-terminal Tyr314. We propose that the formation of the catalytic competent complex of FNR and substrates is achieved through the interaction of the N- and C-terminal segments with ferredoxin and NADP(+), respectively. Since the ends of the polypeptide chain act as flexible regions of proteins, they may contribute to the search of a larger space for a binding partner and to the opening of active sites.

Modified multiplex PCR for identification of Bacillus Calmette-Guérin substrain Tokyo among clinical isolates.

When an adverse reaction occurs and a mycobacterial species is isolated from a person vaccinated with Bacillus Calmette-Guérin (BCG) or a patient receiving BCG immunotherapy, it is essential to identify whether the isolate is BCG or another mycobacterial species. However, differentiation of BCG from other members of Mycobacterium tuberculosis complex has been very difficult. Using several specific primer-pairs, Bedwell et al. [Bedwell J, Kairo SK, Behr MA, Bygraves JA. Identification of substrains of BCG vaccine using multiplex PCR. Vaccine 2001; 19: 2146-51] recently reported that they could distinguish BCG substrains. We modified their method to improve differentiation of Tokyo 172 from other members of the M. tuberculosis complex, and examined whether this modified method could be applied to clinical isolates. Our method clearly identified BCG substrain (BCG Tokyo 172) among clinical isolates and easily distinguished between M. tuberculosis and wild-type Mycobacterium bovis.

Conformational change of H+-ATPase beta monomer revealed on segmental isotope labeling NMR spectroscopy.

F1-ATPase has been shown to be a stepwise molecular motor. Its rotation mechanism has been explained by the interaction of the gamma axis with the open and closed forms of the beta subunit. Although NMR should be a powerful method for elucidating its mechanism, its molecular size (473 amino acid residues, 52 kDa) is a major obstacle. We have applied segmental labeling based on intein ligation to the beta subunit, and succeeded in assigning 89% of the NH (402/451), 89% of the Calpha (417/473), 83% of the Cbeta (357/431), and 90% of the CO (425/473) signals of the beta subunit monomer. The secondary structures predicted from the chemical shifts of the main chain atoms and the relative orientations determined from residual dipolar couplings indicated that the subunit beta monomer takes on the open form in the absence of nucleotide. Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotide binding show that conformational change from the open to the closed form takes place on nucleotide binding. The intrinsic conformational change of the beta subunit monomer induced by nucleotide binding must be one of the essential driving forces for the rotation of F1-ATPase.

A 3-bp deletion mutation of PTPN11 in an infant with severe Noonan syndrome including hydrops fetalis and juvenile myelomonocytic leukemia.

A de novo 3-bp deletion (179-181delGTG) was identified at exon 3 of the PTPN11 gene in a female infant with severe Noonan phenotype including hydrops fetalis and juvenile myelomonocytic leukemia. Since the 3-bp deletion is predicted to result in loss of the 60th glycine in the N-SH2 domain that is directly involved in the intramolecular interaction between the N-SH2 and the PTP domains of the PTPN11 protein, this mutation would disrupt the N-SH2/PTP binding in the absence of a phosphopeptide, leading to an excessive phosphatase activity. The results expand the spectrum of PTPN11 mutations in Noonan syndrome (NS), and suggest that a PTPN11 mutation leads to a wide range of clinical features of Noonan syndrome.

Structure of human PRL-3, the phosphatase associated with cancer metastasis.

PRL-3, a novel class protein of prenylated tyrosine phosphatase, is important in cancer metastasis. Due to its high levels of expression in metastatic tumors, PRL-3 may constitute a useful marker for metastasis and might be a new therapeutic target. Here, we present the solution structure of the phosphatase domain of a human PRL-3 (residues 1-162) in phosphate-free state. The nuclear magnetic resonance (NMR) structure of PRL-3 is similar to that of other known phosphatases with minor differences in the secondary structure. But the conformation and flexibility of the loops comprising the active site differ significantly. When phosphate ions or sodium orthovanadate, which is a known inhibitor, are added to the apo PRL-3, the NMR signals from the residues in the active site appeared and could be assigned, indicating that the conformation of the residues has been stabilized.

[Reliability of bioluminescent assay (ATP method) for testing antimicrobial susceptibility of Mycobacterium tuberculosis].

To evaluate the reliability of our previously reported antimicrobial susceptibility test by ATP method, we have compared our ATP method to the reference test methods such as Mycobacteria Growth Indicator Tube(MGIT) method, minimum inhibitory concentration(MIC) method, NCCLS M24-T agar proportion method(M24-T method), and Vite spectrum method. The concentrations of drugs used for the assessment were isoniazid(INH) 0.1 microgram/ml, rifampicin(RFP) 2.0 micrograms/ml, ethambutol(EB) 2.5 micrograms/ml, streptomycin (SM) 2.0 micrograms/ml, and kanamycin (KM) 5.0 micrograms/ml. When six M. tuberculosis ATCC strains were subjected to 6 independent experiments by using ATP method, highly reproducible results were obtained on the fifth day of the incubation. We examined correlation among ATP method and reference test methods in drug susceptibility testing for 65 clinical isolates of M. tuberculosis. The correlation between ATP method and MGIT-, MIC-, M24-T method were more than 95% for all drugs. When ATP method and Vite spectrum method was compared, the correlation was 87.7% for INH, 98.5% for RFP, 90.8% for EB, 92.3% for SM, 96.9% for KM. The culture period for determining susceptibility between ATP method and MGIT method was compared by using ATCC reference strains and clinical isolates. Six M. tuberculosis ATCC strains were subjected to 6 independent experiments. By the MGIT method, 8 days were required to obtain the results, whereas 3 days were enough by the ATP method. For 65 clinical isolates, the MGIT method required 9 days for determining susceptibility of all isolates. The ATP method required only 5 days for the same strains. These data demonstrate that the improved ATP method that we reported, is simple, rapid, highly reproducible and nonradiometric, and could be used for the assessment of drug susceptibility for M. tuberculosis with high reliability.