Implementation of remote monitoring and diffraction evaluation systems at the Photon Factory macromolecular crystallography beamlines

At the Photon Factory macromolecular crystallography beamlines, two new functions, remote monitoring and diffraction image evaluation, have been developed and installed on the beamline controlling system STARS (simple transmission and retrieval system)

"Per cell" normalization method for mRNA measurement by quantitative PCR and microarrays

BACKGROUND: Transcriptome data from quantitative PCR (Q-PCR) and DNA microarrays are typically obtained from a fixed amount of RNA collected per sample. Therefore, variations in tissue cellularity and RNA yield across samples in an experimental series compromise accurate determination of the absolute level of each mRNA species per cell in any sample. Since mRNAs are copied from genomic DNA, the simplest way to express mRNA level would be as copy number per template DNA, or more practically, as copy number per cell. RESULTS: Here we report a method (designated the "Percellome" method) for normalizing the expression of mRNA values in biological samples. It provides a "per cell" readout in mRNA copy number and is applicable to both quantitative PCR (Q-PCR) and DNA microarray studies. The genomic DNA content of each sample homogenate was measured from a small aliquot to derive the number of cells in the sample. A cocktail of five external spike RNAs admixed in a dose-graded manner (dose-graded spike cocktail; GSC) was prepared and added to each homogenate in proportion to its DNA content. In this way, the spike mRNAs represented absolute copy numbers per cell in the sample. The signals from the five spike mRNAs were used as a dose-response standard curve for each sample, enabling us to convert all the signals measured to copy numbers per cell in an expression profile-independent manner. A series of samples was measured by Q-PCR and Affymetrix GeneChip microarrays using this Percellome method, and the results showed up to 90 % concordance. CONCLUSION: Percellome data can be compared directly among samples and among different studies, and between different platforms, without further normalization. Therefore, "percellome" normalization can serve as a standard method for exchanging and comparing data across different platforms and among different laboratories

UV LED lighting for automated crystal centring

A low-cost light-emitting diode (LED) UV source has been developed for facilitating macromolecular sample centring in the X-ray beam

High-throughput operation of sample-exchange robots with double tongs at the Photon Factory beamlines

Sample-exchange robots with a double-tongs system that could exchange samples within 10 s have been developed

The lysophosphatidic acid receptor LPA(4) regulates hematopoiesis-supporting activity of bone marrow stromal cells

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that acts through G protein-coupled receptors (LPA(1-6)). Although several biological roles of LPA(4) are becoming apparent, its role in hematopoiesis has remained unknown. Here, we show a novel regulatory role for LPA(4) in hematopoiesis. Lpar4 mRNA was predominantly expressed in mouse bone marrow (BM) PDGFR alpha(+) stromal cells, known as the components of the hematopoietic stem/progenitor cell (HSPC) niche. Compared with wild-type mice, LPA(4)-deficient mice had reduced HSPC numbers in the BM and spleen and were hypersusceptible to myelosuppression, most likely due to impairments in HSPC recovery and stem cell factor production in the BM. Analysis of reciprocal BM chimeras (LPA(4)-deficient BM into wild-type recipients and vice versa) indicated that stromal cells likely account for these phenotypes. Consistently, LPA(4)-deficient BM stromal cells showed downregulated mRNA expression of stem cell factor and tenascin-c in vitro. Taken together, these results suggest a critical and novel role for the LPA/LPA(4) axis in regulating BM stromal cells

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 Å resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 Å resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix

Allosteric Modulation of the RNA Polymerase Catalytic Reaction Is an Essential Component of Transcription Control by Rifamycins

SummaryRifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 Å resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation σ factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over ∼19 Å to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal

Risk factors for perioperative venous thromboembolism: A retrospective study in Japanese women with gynecologic diseases

BACKGROUND: Patients with gynecologic cancer have a high risk of venous thromboembolism (VTE) like patients with other cancers. However, there is little information on risk factors for VTE during gynecologic surgery and no uniform preventive strategy. Our objectives were to identify risk factors for perioperative VTE in gynecologic patients and establish methods for prevention. METHODS: We analyzed 1,232 patients who underwent surgery at the Department of Obstetrics and Gynecology of St. Marianna University School of Medicine between January 2005 and June 2008. We investigated (1) risk factors for preoperative VTE, (2) use of an inferior vena cava (IVC) filter, and (3) risk factors for postoperative VTE. RESULTS: There were 39 confirmed cases of perioperative VTE (3.17%), including 25 patients with preoperative VTE and 14 with postoperative VTE. Thirty-two patients had cancer and seven patients had benign diseases. Twenty-two of the 32 cancer patients (68.7%) had preoperative VTE, while postoperative VTE occurred in 10 cancer patients. Multivariate analysis indicated that ovarian cancer, tumor diameter ≥10 cm, and previous of VTE were independent risk factors for preoperative VTE. Among ovarian cancer patients, multivariate analysis showed that an age ≥50 years, the presence of heart disease, clear cell adenocarcinoma, and tumor diameter ≥20 cm were independent risk factors for preoperative VTE. The factors significantly related to preoperative VTE in patients with benign disease included previous VTE, age ≥55 years, tumor diameter ≥20 cm, and a history of allergic-immunologic disease. Thirteen of the 25 patients (52%) with preoperative VTE had an IVC filter inserted preoperatively. Postoperative screening (interview and D-dimer measurement) revealed VTE in 14/1,232 patients (1.14%). Multivariate analysis indicated that cancer surgery, a history of allergic-immunologic disease, and blood transfusion ≥2,000 ml were independent risk factors for postoperative VTE. CONCLUSIONS: Perioperative VTE is often fatal and preventive measures should be taken in the gynecologic field, especially when patients have the risk factors identified in this study. Since VTE is often present before surgery, preoperative screening is important and use of an IVC filter should be considered