Expression of two Trichoderma reesei xylanases in the fission yeast Schizosaccharomyces pombe

Two xylanase genes (xyn1 and xyn2) were amplified by the polymerase chain reaction (PCR) technique from first-strand cDNA prepared from mRNA of Trichoderma reesei QM9414. The genes were located under the human cytomegalovirus gene promoter (CMVp) on copy-number-controlled plasmids (pTLxyn1 and pTLxyn2). When both plasmids were introduced into Schizosaccharomyces pombe, functional xylanases (XYN I and XYN II) were secreted by the recombinant yeasts. The secreted XYN I protein had a molecular mass of 21 kDa whereas XYN II was produced as two molecular forms with sizes of 21 and 28 kDa, the former being not glycosylated and the latter N-glycosylated. XYN I was secreted in the culture medium at a level of about 25 microg/ml and XYN II at about 170 microg/ml. The recombinant xylanases had the same characteristics with respect to the effects of temperature and pH on the enzyme activity as the native ones.     

The protein encoded by cancer/testis gene D40/AF15q14 is localized in spermatocytes, acrosomes of spermatids and ejaculated spermatozoa

We have previously identified and cloned a human gene, D40, that is preferentially expressed in testis among normal organs, while it is widely expressed in various human tumor cell lines and primary tumors derived from different organs. In this report, we have examined the expression and localization of this protein in human testis with an antibody specific to D40 protein. In Western analyses, the anti-D40 antibody recognized a major band with a molecular mass of 300 kDa and a minor band of 250 kDa. These bands were not observed in the testis lysates from patients with Sertoli-cell-only syndrome and with Kleinfelter syndrome, who lack germ cells of the testis, indicating that D40 protein is expressed in the germ cells of normal testis. Immunohistochemical studies have revealed that D40 protein is highly expressed in spermatocytes and in the pre-acrosome of round spermatids. In the acrosome, D40 protein expression is observed not inside but outside the acrosome membrane. This is consistent with the finding that the amino-acid sequence at the amino terminal of the D40 protein lacks a hydrophobic signal peptide that is required for proteins to translocate to the membrane. Expression of D40 protein is observed in the acrosome of ejaculated spermatozoa as well, although the level is low compared with that in the pre-acrosome of spermatids. These results suggest that D40 protein plays important roles in spermatogenesis, especially in the formation and maintenance of the acrosome.     

Radiation doses to neonates during X ray computed tomography examinations

As the survival rate of newborns has increased, the number of X ray computed tomography (CT) examinations performed on neonates has been increasing. The exposure doses from CT examinations are known to be higher than those from conventional radiography. Although radiation sensitivity of neonates is higher than that of adults, there are few reports on dose estimates of neonates in CT examinations. Four cylindrical phantoms and one neonatal phantom have been developed to estimate doses to neonates during CT examinations. Using these phantoms and glass dosemeters, absorbed doses were measured. Estimated exposure doses to neonates were higher than those to adults, and our results suggest a need to optimise carefully CT examinations in newborns.     

Determination of captafol,cyhexatin, 1-naphthylacetic acid and quintozene in apple,Japanese pear and melon by simultaneous extraction

A simple and rapid method is described for the determination of the non-registered pesticides, captafol, quintozene (PCNB), cyhexatin and 1-naphthylacetic acid (NAA), in fruits. These pesticides were extracted with acidified acetone, then captafol and PCNB were purified with a Florisil mini column and analyzed by GC-ECD. Cyhexatin was ethylated with ethylmagnesium bromide, and the ethyl derivative was analyzed by GC-FPD (Sn filter). NAA was purified with liquid-liquid extraction and determined by HPLC equipped with a fluorescence detector. These analytes were identified with GC/MS or LC/MS. The minimum identified concentration of the pesticides was below 0.2 ng per injection, which corresponds to a detection limit of below 0.02 microgram/g in the original samples. Recoveries of the pesticides spiked at 0.1 microgram/g into apple, Japanese pear and melon were greater than 61%.     

Real Space Imaging of the Electronic States in Underdoped Ca2−x Na x CuO2Cl2 Single Crystals

Scanning tunneling microscopy (STM) has been performed on underdoped Ca_2–x Na _x CuO₂Cl₂ (Na-CCOC) single crystals to investigate the electronic states of doped Mott insulators near the metal-insulator transition. STM images taken at 7 K show patch-like or river-like irregular features superposed on the atomic corrugations. The irregular structure has a characteristic length scale of 20 Å, which is unchanged in different samples and at different doping levels studied (0.08<x<0.12). Bias voltage dependence of the STM image suggests that Na-CCOC consists of two distinct phases with different electronic states.     

Computer-aided design of a factor Xa inhibitor by using MCSS functionality maps and a CAVEAT linker search

We have investigated a new approach to efficiently find a novel inhibitor against a serine protease (i.e. an activated coagulation factor X, FXa) by using de novo design programs and the X-ray crystal structure of the target enzyme. FXa is a coagulant enzyme that generates thrombin (a serine protease) and participates in both intrinsic and extrinsic coagulation pathways. We adopted multiple copy simultaneous search (MCSS) and CAVEAT linker search techniques, which disclosed a novel FXa inhibitor (T01312) consisting of two binding moieties (the benzamidinyl and adamantyl groups) and a linker unit (the carboxybenzylamine group). The inhibitory activity of T01312 against FXa was determined to be a small K(i)-value of 48nM, which is two orders of magnitude smaller than that against thrombin. An X-ray crystal analysis of T01312 complexed with trypsin (an analogue of FXa) and docking studies of T01312 with trypsin and FXa showed that: (i) the benzamidinyl group is a predominant binding moiety in the anionic pocket (S1 site) with an asparatic acid residue; (ii) a hydrophobic pocket (S4 site) is the binding site of the adamantyl group; (iii) the carboxylate group of the linker contributes to the selectivity for FXa against thrombin. Thus, the combination of the knowledge of the X-ray crystal structure of the target molecule with MCSS and CAVEAT linker search techniques proved to be an effective hit-finding method that does not require the screening of huge compound libraries.     

Numerical prediction of sound generated from flows with a low Mach number

Numerical computations of sound generated from flows with a low Mach number are presented based on Lighthill’s acoustic analogy with an assumption that sound does not alter the flow field from which it is generated. The source fluctuations of the flow field are computed by a large-eddy simulation (LES) with Dynamic Smagorinsky Model (DSM) and they are fed to the following acoustical computation as input data. An explicit/implicit finite element method with second order accuracy both in time and space is used for flow field discretization. The method is applied to the prediction of sound in three different classes of problems: far-field sound generated from flow around a bluff body, sound resulting from blade-stator interaction of turbomachinery and sound due to a turbulent boundary layer on an aerofoil. The computed frequency spectra of the sound show a fairly good agreement with the measured spectra for all the cases.     

An augmented approach for Stokes equations with a discontinuous viscosity and singular forces

For Stokes equations with a discontinuous viscosity across an arbitrary interface or/and singular forces along the interface, it is known that the pressure is discontinuous and the velocity is non-smooth. It has been shown that these discontinuities are coupled together, which makes it difficult to obtain accurate numerical solutions. In this paper, a new numerical method that decouples the jump conditions of the fluid variables through two augmented variables has been developed. The GMRES iterative method is used to solve the Schur complement system for the augmented variables that are only defined on the interface. The augmented approach also rescales the Stokes equations in such a way that a fast Poisson solver can be used in each iteration. Numerical tests using examples that have analytic solutions show that the new method has average second order accuracy for the velocity in the infinity norm. An example of a moving interface problem is also presented.