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The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol−1) to the exergonic reduction of H+ by reduced ferredoxin (Fdred) (∆G0 ≈ − 16 kJ mol−1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H2)-dependent reduction of nicotinamide adenine dinucleotide (NAD+) in the presence of oxidized ferredoxin (Fdox) at a rate of ≈17 μmol NADH min−1 mg−1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases. [ABSTRACT FROM AUTHOR]

Purpose: To clarify the profile of adverse events from endocrine therapies in older patients.Methods: We surveyed 15 subjective symptoms including hot flashes, sweating, knuckle stiffness, knee/shoulder joint pain, limb numbness, lethargy, forgetfulness, depressive state, irritated state, genital bleeding, leukorrhea increase, vaginal dryness, bone fracture, and weight gain by a questionnaire among 2044 patients over 55 years old (total number of answered sheets, 8875) and compared the results according to age (56-69 years old vs. ≥ 70 years old) and type of therapy (aromatase inhibitors (AIs) vs. selective estrogen receptor modulators (SERMs)). Among patients 56-69 years old, 6093 and 314 responses were from patients treated with AIs (1477 patients) and SERMs (123 patients), respectively, and 2292 and 176 responses were from those ≥ 70 years old treated with AIs (581 patients) and SERMs (51 patients), respectively.Results: In patients ≥ 70 years old, sweating, knuckle stiffness, knee/shoulder joint pain, limb numbness, and lethargy were significantly more frequent/severe with AIs than with SERMs. In those aged 56-69, knuckle stiffness and vaginal dryness were significantly more frequent with AIs than with SERMs, but the opposite occurred for hot flashes, leukorrhea increase, genital bleeding, and weight gain.Conclusions: Among patients ≥ 70 years old, many symptoms were significantly more frequent/severe with AIs than with SERMs, compared with those aged 56-69, which suggests a difference in the profile of adverse events according to the type of endocrine therapy and the patient's age. It is important to consider the benefits and risks of each treatment to optimize endocrine therapy for older patients. [ABSTRACT FROM AUTHOR]

Eight years after surgical treatment for breast cancer, serum p53 antibody levels suddenly increased in a 67-year-old woman without recurrent signs of cancer. Early esophageal squamous cell carcinoma was detected by endoscopic screening. After endoscopic submucosal dissection was completed, serum p53 antibody levels decreased to be in the normal range. Serum p53 antibody monitoring can be helpful for early detection of not only recurrence of primary carcinoma but also development of metachronous carcinoma. This case is the first to monitor the changing titers of serum p53 antibody at the early phase of esophageal cancer progression into lamina propria mucosae until the disappearance of tumor cells. [ABSTRACT FROM AUTHOR]

The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H−) bridging the Ni and Fe and a proton (H+) attached to the sulphur of a cysteine ligand. The Ni-H− and Fe-H− bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN− ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis. [ABSTRACT FROM AUTHOR]

The [NiFe] hydrogenase from the sulphate-reducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SIr, provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SIa, Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SIr was found to be light-sensitive ( T ≤ 110 K), similar to the active Ni-C and the CO-inhibited states. Transition to the final photoproduct state (Ni-SL) was shown to involve an additional transient light-induced state (Ni-SI1961). Rapid scan kinetic infrared measurements provided activation energies for the transition from Ni-SL to Ni-SIr in protonated as well as in deuterated samples. The inhibitor CO was found not to react with the active site of the Ni-SL state. The wavelength dependence of the Ni-SIr photoconversion was examined in the range between 410 and 680 nm. Light-induced effects were associated with a nickel-centred electronic transition, possibly involving a change in the spin state of nickel (Ni2+). In addition, at T ≤ 40 K the CN− stretching vibrations of Ni-SL were found to be dependent on the colour of the monochromatic light used to irradiate the species, suggesting a change in the interaction of the hydrogen-bonding network of the surrounding amino acids. A possible mechanism for the photochemical process, involving displacement of the oxygen-based ligand, is discussed. [ABSTRACT FROM AUTHOR]

Periostin is a secreted protein that shares structural homology with the insect axon guidance protein fasciclin 1. Periostin is expressed predominantly in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses. We have shown previously that periostin is a novel component of subepithelial fibrosis in bronchial asthma. Here, we investigated the relationship between periostin and bone marrow (BM) fibrosis. Periostin was expressed in the stroma and stromal cells of BM fibrosis specimens and to a great extent its expression levels correlated closely to the grade of fibrosis, as estimated by silver staining. However, in the present study, we found no relationship between plasma periostin levels and the extent of BM fibrosis. We also demonstrated that periostin is secreted by human BM hTERT stromal cells and that its secretion is enhanced by TGF-beta, a cytokine produced by clonal proliferation of megakaryocytes and/or monocytes. These results indicate that periostin is a component of BM fibrosis and that it may play a role in the disease progression. [ABSTRACT FROM AUTHOR]

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH− bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme. [ABSTRACT FROM AUTHOR]

Approximately 20% of mantle cell lymphomas (MCL) present with the blastoid variant associated with poor prognosis. Lactic acidosis complicated with hematologic malignancies is seen infrequently and is associated with a poor outcome. Here we report the case of a patient with the blastoid variant of MCL complicated by lactic acidosis and who achieved complete remission through chemotherapy combined with rituximab therapy. A 77-year-old man presented with peripheral blood lymphoma cells, huge splenomegaly, abdominal and mediastinal lymphadenopathy, and pleural effusion. A bone marrow smear showed an increase in large, abnormal lymphoid cells with oval or round nuclei, distinct nucleoli, and abundant basophilic cytoplasm with vacuolization. Splenic sections also showed massive and diffuse infiltration by these cells. Flow cytometry analysis showed these cells to be positive for CD5, CD19, CD20, and kappa chain and negative for CD10 and CD23. A blastoid variant of MCL was diagnosed from the results of histologic, immunohistochemical (cyclin D1), and cytogenetic (chimeric bcl-1/IgH fusion gene) analyses. The patient recovered from the 2 episodes of severe lactic acidosis for which he had been given chemotherapy, and he achieved complete remission after subsequent chemotherapy combined with rituximab treatment. [ABSTRACT FROM AUTHOR]

Annexin V is a Ca2+-dependent phospholipid binding protein. Although it has been shown to inhibit protein kinase C (PKC) in cell-free systems, its role in the intact cell is unclear. A stable MCF-7 human breast cancer cell overexpression system was established to investigate the function of annexin V. In these cells, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation and kinase activity of ERK1/2 were suppressed. Morphological changes induced by TPA were reduced by annexin V overexpression as well as by the pan-PKC inhibitor, bisindolylmaleimide I, and by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor, PD98059. TPA-induced MEK1/2 and Raf-1 phosphorylation were reduced in these cells. The TPA-enhanced active Ras, and its association with Raf-1, were reduced. TPA treatment of MCF-7 cells caused an increased association of Shc with Grb2. However, this increased association was prevented in the annexin V-overexpressors. p21WAF/CIP1 is responsible for inhibition of cell cycle progression in MCF-7 cells. TPA induced the expression of p21WAF/CIP1 to a greater extent in MCF-7 parent and control plasmid cells than in annexin V overexpressors. PD98059 inhibited this increase, suggesting that TPA upregulation of p21WAF/CIP1 occurs via the MEK pathway, and that annexin V overexpression blunts it. This work shows that annexin V overexpression suppresses the TPA-induced Ras/ERK signaling by inhibiting at/or upstream of Shc, possibly through the inhibition of PKCs. Oncogene (2000) 19, 2904–2912 [ABSTRACT FROM AUTHOR]

Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved. Changes in S-isotope ratios over time provide clues to understanding the co-evolution of Earth and its biosphere. Here the authors determine the isotope effect of the first reductive enzyme in the sulfate respiration pathway and reinterpret sedimentary S-isotope records based on this biochemical constraint. [ABSTRACT FROM AUTHOR]