Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.     

Acetyl-seryl-aspartyl-lysyl-proline is a novel natural cell cycle regulator of renal cells

A natural tetrapeptide, acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a physiological negative regulator of hematopoiesis. The precursor of AcSDKP, thymocin beta 4, is expressed in many tissues including kidney. The present study examined the antiproliferative effect of AcSDKP in two renal cell lines, namely, renal interstitial fibloblasts cell line (NRK 49F) and renal proximal tubular epitherial cells (LLC-PK1). An addition of AcSDKP for 48 hours in theses cells resulted in a concentration-dependent attenuation in the proliferation rate (significant difference to non-treated cells was observed at 10(-9) to 10(-5) M AcSDKP) determined by a colorimetry of alamer blue oxidation. The cell cycle analysis of NRK 49F cells treated with AcSDKP showed that AcSDKP significantly reduced the ratio of S-phase to G2/M-phases. Thus, physiological concentrations of AcSDKP is capable of altering cell cycle to inhibit the proliferation of renal cells.     

Evaluation of two-dimensional electronic portal imaging device using integrated images during volumetric modulated arc therapy for prostate cancer

BackgroundThe aim of the study was to evaluate analysis criteria for the identification of the presence of rectal gas during volumetric modulated arc therapy (VMAT) for prostate cancer patients by using electronic portal imaging device (EPID)-based in vivo dosimetry (IVD).Materials and methodsAll measurements were performed by determining the cumulative EPID images in an integrated acquisition mode and analyzed using PerFRACTION commercial software. Systematic setup errors were simulated by moving the anthropomorphic phantom in each translational and rotational direction. The inhomogeneity regions were also simulated by the I’mRT phantom attached to the Quasar phantom. The presence of small and large air cavities (12 and 48 cm³) was controlled by moving the Quasar phantom in several timings during VMAT. Sixteen prostate cancer patients received EPID-based IVD during VMAT.ResultsIn the phantom study, no systematic setup error was detected in the range that can happen in clinical (< 5-mm and < 3 degree). The pass rate of 2% dose difference (DD2%) in small and large air cavities was 98.74% and 79.05%, respectively, in the appearance of the air cavity after irradiation three quarter times. In the clinical study, some fractions caused a sharp decline in the DD2% pass rate. The proportion for DD2% < 90% was 13.4% of all fractions. Rectal gas was confirmed in 11.0% of fractions by acquiring kilo-voltage X-ray images after the treatment.ConclusionsOur results suggest that analysis criteria of 2% dose difference in EPID-based IVD was a suitable method for identification of rectal gas during VMAT for prostate cancer patients.     

Gait Variability before Surgery and at Discharge in Patients Who Undergo Total Knee Arthroplasty: A Cohort Study

This study aimed to determine gait ability at hospital discharge in patients undergoing total knee arthroplasty (TKA) as an indicator of the risk of falling. Fifty-seven patients undergoing primary TKA for knee osteoarthritis participated in this study. Gait variability measured with accelerometers and physical function including knee range of motion (ROM), quadriceps strength, walking speed, and the Timed Up and Go (TUG) test were evaluated preoperatively and at discharge from the hospital (1 month before and 5 days after surgery). All patients were discharged directly home at 5 days after surgery. Knee flexion of ROM, quadriceps strength, walking speed, and the TUG test results were significantly worse at hospital discharge than preoperatively (p < 0.001). However, gait variability was not significantly different before and after TKA. This result indicated that patients following TKA surgery could walk at hospital discharge as stably as preoperatively regardless of the decrease in physical function, including knee ROM, quadriceps strength, and gait speed after surgery.     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

Surgical Ablation for Atrial Fibrillation in Cardiac Surgery: A Consensus Statement of the International Society of Minimally Invasive Cardiothoracic Surgery (ISMICS) 2009

OBJECTIVE:: This purpose of this consensus conference was to determine whether surgical atrial fibrillation (AF) ablation during cardiac surgery improves clinical and resource outcomes compared with cardiac surgery alone in adults undergoing cardiac surgery for valve or coronary artery bypass grafting. METHODS:: Before the consensus conference, the consensus panel reviewed the best available evidence, whereby systematic reviews, randomized trials, and nonrandomized trials were considered in descending order of validity and importance. Evidence-based statements were created, and consensus processes were used to determine the ensuing recommendations. The American Heart Association/American College of Cardiology system was used to label the level of evidence and class of recommendation. RESULTS:: The consensus panel agreed on the following statements in patients with AF undergoing cardiac surgery concomitant surgical ablation: CONCLUSIONS:: Given these evidence-based statements, the consensus panel stated that, in patients with persistent and permanent AF undergoing cardiac surgery, concomitant surgical ablation is recommended to increase incidence of sinus rhythm at short- and long-term follow-up (class 1, level A); to reduce the risk of stroke and thromboembolic events (class 2a, level B); to improve EF (class 2a, level A); and to exercise tolerance (class 2a, level A) and long-term survival (class 2a, level B).     

Critical function of death-associated protein 3 in T cell receptor-mediated apoptosis induction

Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.     

Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.     

Cytological changes during cell wall sac formation in mulberry idioblasts

Summary. When calcium carbonate crystals are formed in mulberry (Morus abla) idioblasts, they are deposited in newly formed cell wall sacs. The initial cytological events leading to cell wall sac formation were observed in the distal end of young idioblasts and tentatively categorized into four stages. The first indication of formation was the separation of the innermost cell wall layer from the cell wall, which is followed by the deposition of egg-shaped polysaccharide on the inner cell wall surface. The size of the deposit area increased, while the thickness of the cell wall significantly decreased during the next stage. Finally, the condensed cellulosic lamella was invaginated into the deposition area, resulting in the formation of an elongated cell wall sac. The internal cell wall sac was composed of numerous fibers with different morphologies. Application of gelatin-methenamine-silver staining allowed us to observe the spatial distribution of cellulosic polysaccharides as electron-dense images. Correspondence and reprints: Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.