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Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2‐sensitive [NiFe]‐hydrogenases. Although the activities of some O2‐tolerant [NiFe]‐hydrogenases are unaffected by CO, the partially O2‐tolerant [NiFe]‐hydrogenase from Citrobacter sp. S‐77 (S77‐HYB) is inhibited by CO. In this work, the CO‐bound state of S77‐HYB was characterized by activity assays, spectroscopic techniques and X‐ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier‐transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni–Fe active site of S77‐HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]‐hydrogenases. [ABSTRACT FROM AUTHOR]

Because the production of tumor‐associated antibodies (TAA) is a humoral immune response in cancer patients, serum autoantibodies may be detected even in patients with early‐stage tumors. Seventeen recombinant proteins with tags in Escherichia coli (p53, RalA, p90, NY‐ESO‐1, HSP70, c‐myc, galectin‐1, Sui1, KN‐HN‐1, HSP40, PrxVI, p62, cyclin B1, HCC‐22‐5, annexin II, HCA25a, and HER2) were applied as capturing antigens in sandwich ELISA to measure serum IgG levels. Sera from 73 healthy donors and 386 patients with breast cancer, including 182 stage 0/I patients, were evaluated using cutoff values for each TAA equal to the mean +3 SD of the serum levels of healthy controls. The positive TAA rates were relatively high for p53 (10%) and RalA (10%). The positive rates of all TAA of stage 0/I were similar to those of all patients. Even in the stage 0/I patients, 24% showed that two or more TAA were positive, and the positive rate of a five‐TAA combination assay was 37%. The positivity rate was significantly higher for the non–luminal type than for the luminal type (P =.003). Logistic analysis showed that seropositivity (positive for one or more TAA) in breast cancer patients was independent from any TNM factor or disease stage and was significantly associated with histological grade in the multivariate analysis (P =.007). TAA in breast cancer patients may be useful for early detection. However, seropositivity of breast cancer reflects the tumor characteristics but not the disease stage. [ABSTRACT FROM AUTHOR]

Appropriate biomarkers are required to predict the clinical outcome of triple‐negative breast cancer (TNBC). In this study, we focused on the clinical importance of two representative tumor‐associated proteins, Bcl‐2 and p53. Bcl‐2 expression is usually related to estrogen receptor expression and a favorable outcome in breast cancer. TNBC has been reported to show a high frequency of p53 positivity suggesting TP53 mutations. The expressions of Bcl‐2 and p53 were immunohistochemically examined in TNBC involving two age groups of postmenopausal women (≥75 y/o, n = 75; 55–64 y/o, n = 47), who underwent surgery without neoadjuvant therapy. We examined their associations with each other, or with clinicopathological factors including the outcome. Bcl‐2 expression was inversely correlated with androgen receptor, apocrine morphology, and p53 expressions, and was an independent predictor of a poor outcome in total or in younger women. p53 positivity was associated with a more favorable outcome than p53 negativity in the younger group. In combined analyzes, none of the twenty Bcl‐2‐negative/p53‐positive cases in the younger group exhibited recurrence, resulting in the independent favorable predictive value of Bcl‐2‐negative/p53‐positive. The anti‐apoptotic nature of Bcl‐2 may be apparent in TNBC. The excellent outcome of Bcl‐2‐negative/p53‐positive cases in the younger group warrants further combined investigation of Bcl‐2/p53 in TNBC. [ABSTRACT FROM AUTHOR]

Direct spectroscopic evidence for a hydride bridge in the Ni-R form of [NiFe] hydrogenase has been obtained using iron-specific nuclear resonance vibrational spectroscopy (NRVS). The Ni-H-Fe wag mode at 675 cm−1 is the first spectroscopic evidence for a bridging hydride in Ni-R as well as the first iron-hydride-related NRVS feature observed for a biological system. Although density function theory (DFT) calculation assisted the determination of the Ni-R structure, it did not predict the Ni-H-Fe wag mode at ∼675 cm−1 before NRVS. Instead, the observed Ni-H-Fe mode provided a critical reference for the DFT calculations. While the overall science about Ni-R is presented and discussed elsewhere, this article focuses on the long and strenuous experimental journey to search for and experimentally identify the Ni-H-Fe wag mode in a Ni-R sample. As a methodology, the results presented here will go beyond Ni-R and hydrogenase research and will also be of interest to other scientists who use synchrotron radiation for measuring dilute samples or weak spectroscopic features. [ABSTRACT FROM AUTHOR]

Membrane type 1 matrix metalloproteinase (MT1-MMP) belongs to the large family of zinc-dependent endopeptidases termed MMPs that are located in the extracellular matrix. MT1-MMP was crystallized at 277 K using the vapour-diffusion method with PEG as a precipitating agent. Data sets for MT1-MMP were collected to 2.24 Å resolution at 100 K. The crystals belonged to space group P43212, with unit-cell parameters a = 62.99, c = 122.60 Å. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient ( VM) of 2.90 Å3 Da−1; the solvent content is estimated to be 57.6%. [ABSTRACT FROM AUTHOR]

Nuclear inelastic scattering of 57Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign FeCO and FeCN bending and stretching vibrations of the active site outside the spectral range of the FeS cluster normal modes. [ABSTRACT FROM AUTHOR]

Blick ins Zentrum: Die Methode der inelastischen kernresonanten Streuung (NRVS) wurde genutzt, um 57Fe ‐ markierte [NiFe] ‐ Hydrogenase in unterschiedlichen Zuständen des Enzyms zu untersuchen. Damit gelang es, die Fe ‐ CO ‐ und Fe ‐ CN ‐ Beuge ‐ und ‐ Streckschwingung außerhalb des Spektralbereichs der Normalmoden der Fe ‐ S ‐ Cluster zu detektieren und zuzuordnen (siehe Spektrum). [ABSTRACT FROM AUTHOR]

We report two cases in which visualization of the segmental branch of the hepatic portal vein with the colour Doppler ultrasonography (US) technique was useful for the early diagnosis of veno-occlusive disease. The change in blood flow in the segmental branch of the portal vein occurred 5 and 6 d before the clinical criteria were fulfilled in the two cases. Reverse flow in the segmental branch began partially in the liver at first, and then spread to the whole liver several days later. All the US findings in both cases disappeared after thrombolytic therapy. [ABSTRACT FROM AUTHOR]

Dissimilatory sulfite reductase (Dsr) plays an important role in sulfate respiration in many sulfate-reducing bacteria. Dsr from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the sitting-drop vapour-diffusion method with PEG 3350 and potassium thiocyanate as precipitants. A data set was collected to 3.7 Å resolution from a single crystal at 100 K using synchrotron radiation. The Dsr crystal belonged to space group P41212, with unit-cell parameters a = b = 163.26, c = 435.32 Å. The crystal structure of Dsr was determined by the molecular-replacement method based on the three-dimensional structure of Dsr from D. vulgaris Hildenborough. The crystal contained three α2β2γ2 units per asymmetric unit, with a Matthews coefficient ( VM) of 2.35 Å3 Da−1; the solvent content was estimated to be 47.7%. [ABSTRACT FROM AUTHOR]

Ribonucleotide reduction, the unique step in DNA-precursor biosynthesis, involves radical-dependent redox chemistry and diverse metallo-cofactors. The metallo-cofactor (R2F) encoded by the nrdF ( nucleotide re duction) gene in Corynebacterium ammoniagenes ATCC 6872 was isolated after homologous expression and a new crystal form of ribonucleotide reductase R2F was obtained. R2F was crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. A data set was collected to 1.36 Å resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group C2, with unit-cell parameters a = 96.21, b = 87.68, c = 83.25 Å, β = 99.29°. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient ( VM) of 2.69 Å3 Da−1; the solvent content was estimated to be 54.3%. X-ray fluorescence spectroscopy and MAD diffraction data indicated the presence of manganese in the molecule and the absence of iron. [ABSTRACT FROM AUTHOR]

Light-oxygen-voltage (LOV) proteins play an important role in blue-light-dependent physiological processes in many organisms. The LOV domain of the blue-light receptor YtvA from Bacillus amyloliquefaciens FZB42 has been purified and crystallized at 277 K using the sitting-drop vapour-diffusion method with 2-ethoxyethanol as a precipitant. A data set was collected to 1.60 Å resolution from a single crystal at 100 K using synchrotron radiation. The LOV domain of YtvA crystallized in space group C2221, with unit-cell parameters a = 64.95, b = 83.76, c = 55.81 Å. The crystal structure of the LOV domain of YtvA was determined by the molecular-replacement method. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient ( VM) of 3.04 Å3 Da−1; the solvent content was estimated to be 59.5%. [ABSTRACT FROM AUTHOR]

Sulfur in its various oxidation states is used for energy conservation in many microorganisms. Adenylylsulfate reductase is a key enzyme in the sulfur-reduction pathway of sulfate-reducing bacteria. The adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the vapour-diffusion method with ammonium sulfate as the precipitating agent. A data set was collected to 1.7 Å resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group P31, with unit-cell parameters a = b = 125.93, c = 164.24 Å. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient ( VM) of 4.02 Å3 Da−1; the solvent content was estimated to be 69.4%. [ABSTRACT FROM AUTHOR]

Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of β-catenin by glycogen synthase kinase 3β. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P61 or P65, with unit-cell parameters a = b = 91.49, c = 84.92 Å. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 Å. [ABSTRACT FROM AUTHOR]

The catalytic domain of death-associated protein kinase (DAPK) has been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with PEG 8000 and magnesium acetate as precipitants. Complexes with the inhibitor staurosporine and its analogue BDB402 were also crystallized in the presence of PEG 400 and PEG 8000, respectively. Diffraction data were collected to 2.4 Å for the native catalytic domain, to 2.9 Å for the staurosporine complex and to 2.7 Å for the BDB402 complex. All three crystals belong to space group P212121, with unit-cell parameters a = 77.992, b = 109.909, c = 50.063 Å for the catalytic domain, a = 78.911, b = 113.162, c = 50.658 Å for the staurosporine complex and a = 77.337, b = 108.869, c = 50.186 Å for the BDB402 complex. In both complexes the inhibitor molecule was clearly assigned in the difference Fourier map calculated on the basis of the phases obtained from the structure of the catalytic domain. [ABSTRACT FROM AUTHOR]

A new perspective on enzymes: Free‐electron lasers are increasingly used to obtain crystal structures of interesting enzymes like photosystem II from nanocrystals at room temperature, to avoid radiation damage, and to detect structural differences between specific states in the catalytic cycle. [ABSTRACT FROM AUTHOR]

Neuer Blick auf Enzyme: Freie ‐ Elektronen ‐ Laser werden zunehmend genutzt, um Strukturen von Nanokristallen interessanter Enzyme wie dem Photosystem II bei Raumtemperatur aufzuklären, Strahlungsschäden zu vermeiden und Strukturunterschiede spezifischer Zustände während der Katalyse zu detektieren. [ABSTRACT FROM AUTHOR]

The active site of [NiFe] hydrogenase is an excellent catalyst for hydrogen conversion with an intriguing ability to recover from exposure to dioxygen. In their Communication on page 724 ff., W. Lubitz, S. P. Cramer, and co‐workers use the synchrotron technique of nuclear resonance vibrational spectroscopy to identify normal modes associated with active‐site FeCN and FeCO motion. [ABSTRACT FROM AUTHOR]

Das aktive Zentrum der [NiFe] ‐ Hydrogenase ist ein exzellenter Katalysator der Wasserstoffumsetzung mit der faszinierenden Fähigkeit, sich nach Sauerstoffexposition zurückzubilden. W. Lubitz, S. P. Cramer et al. nutzen in ihrer Zuschrift auf S. 752 ff. die Synchrotrontechnik der kernresonanten inelastischen Streuung, um Normalmoden der Fe ‐ CN ‐ und Fe ‐ CO ‐ Bewegung im aktiven Zentrum zu identifizieren. [ABSTRACT FROM AUTHOR]

Nitrophorins (nitric oxide transport proteins) are haemproteins originating from the blood-feeding insect Rhodnius prolixus. They consist of an eight-stranded β-barrel, which classifies them into the lipocalin family. Nitrophorin 7 (NP7) and the E27V mutant protein NP7(E27V) were crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. Data sets for wild-type NP7 and NP7(E27V) were collected to 1.80 Å resolution from single crystals at 100 K using synchrotron radiation. The crystals belonged to space group P2(1), with unit-cell parameters a = 38, b = 67, c = 39 Å, β = 117°. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (V(M)) of 2.11 Å(3) Da(-1); the solvent content was estimated to be 41.8%., (© 2012 International Union of Crystallography. All rights reserved.)

The membrane-bound [NiFe] hydrogenase is a unique metalloprotein that is able to catalyze the reversible oxidation of hydrogen to protons and electrons during a complex reaction cycle. The [NiFe] hydrogenase was isolated from the photosynthetic purple sulfur bacterium Allochromatium vinosum and its crystallization and preliminary X-ray analysis are reported. It was crystallized by the hanging-drop vapour-diffusion method using sodium citrate and imidazole as crystallization agents. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 205.00, b = 217.42, c = 120.44 A. X-ray diffraction data have been collected to 2.5 A resolution.

The F-box protein Skp2 positively regulates the G1-S transition by promoting degradation of the cyclin-dependent kinase inhibitor p27(kip1) (p27). Recent evidence has suggested an oncogenic role of Skp2 in not only carcinogenesis but also lymphomagenesis. In this study, we performed immunohistochemical analysis on the cell-cycle-associated proteins, Skp2, p27, and Ki-67, in 27 patients with de novo diffuse large B-cell lymphoma (DLBCL), evaluating the correlation between the clinical characteristics and expression levels of these proteins. The patients were classified into two groups according to the positivity for Skp2 expression: a high Skp2 expression group (>60% positive for Skp2 in lymphoma cells) and a low Skp2 expression group (< or = 60%). A high level of Skp2 expression significantly correlated with advanced clinical stage (P = 0.029), although the increase did not correlate with age, gender, LDH levels, presence of extranodal disease, or performance status and resulted in no correlation with the International Prognostic Index-based risk grading. However, it was noteworthy that the high Skp2 expression group demonstrated a significantly worse prognosis than the low Skp2 expression group (P = 0.0007). The expression level of Skp2 correlated with that of Ki-67 but not necessarily with that of p27. The p27 expression level did not correlate patients' prognosis. Taken together, it was suggested that Skp2 was a valuable and independent marker predicting the outcome in DLBCL., (Copyright 2003 Wiley-Liss, Inc.)