Classification of Clostridium butyricum based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and pulsed-field gel electrophoresis

Eleven strains of Clostridium butyricum collected from different sources were analysed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and pulsed-field gel electrophoresis (PFGE). The strains could be classified into four groups based on their banding profiles of the proteins extracted from the cells on SDS-PAGE. Group I consisted of seven strains, and these strains were further divided into five subgroups by PFGE. The strains belonging to groups II, III, and IV on SDS-PAGE were also classified into the same II to IV groups by PFGE. These data indicate that grouping of the strains of C. butyricum can be performed by employing both SDS-PAGE and PFGE.     

Ultrastructural aspects of leaves inFestuca ovina andPoa sphondylodes (C-3 Poaceae)

Structural aspects of the leaves of two common festucoids,Festuca ovina andPoa sphondylodes, have been examined employing the electron microscopy. The nature of vascular bundles and of sheaths that surround vascular tissues was discussed in the study. The festucoids exhibited a non-Kranz C-3 anatomy with more than four mesophyll cells separating the bundle sheaths of a leaf blade. Vascular tissues in theseFestuca andPoa leaves were surrounded by a double sheath: an inner distinct mestome sheath (MST) and an outer indistinctive layer of parenchymatous bundle sheath (PBS) cells. The PBS cells were much larger than the MST and had thin walls. The MST cells were relatively small and rectangular inP. sphondylodes and more or less hexangular in transverse sections ofF. ovina. InP. sphondylodes, MST had conspicuously thickened inner tangential walls with asymmetrically uninterrupted suberized lamellae in radial and tangential walls. In most differentiated MST cells, all walls were highly suberized. During suberin deposition, MST cells were quite vacuolated and most of the cytoplasm was present as a thin peripheral layer. However, MST walls inF. ovina revealed very thin suberized lamellae with translucent striations. No chloroplasts were detected inP. sphondylodes, whereas the MST inF. ovina contained small chloroplasts. Plasmodesmata were well developed in the primary pit fields of walls between MST and vascular cells, and between adjacent MST cells. Plasmodesmata were less frequent in the walls between the inner and outer sheath cells. Suberized lamellae were totally absent from the PBS cell walls in all veins. External to the PBS, the mesophyll comprised thin walled cells with abundant intercellular spaces. Peripherally arranged chloroplasts in the mesophyll were numerous and often larger than those of PBS and MST cells. Characteristics associated with C-3 and other ultrastructural features were also discussed in the study.     

Anti-(glioma surface antigen) monoclonal antibody G-22 recognizes overexpressed CD44 in glioma cells

We raised an anti-glioma monoclonal antibody, named G-22, that specifically recognizes a human gliomaassociated surface antigen. Proven to be useful for target imaging of malignant gliomas after radioisotope labeling and cerebrospinal fluid diagnosis by enzyme-linked immunospecific assay, G-22 was found to immunoprecipitate an 83-kDa glycoprotein of the human glioma U-251MG cell. We purified this antigen by G-22-coupled cyanogenbromide-activated Sepharose affinity chromatography, and sequence analysis demonstrated that the 54 amino acid residues were identical to positions 55–108 of human CD44. The results show that the smallest spliced form (85 kDa) of CD44 is strongly expressed in glioma cells.     

Accelerated failure time modeling via nonparametric mixtures

An accelerated failure time (AFT) model assuming a log-linear relationship between failure time and a set of covariates can be either parametric or semiparametric, depending on the distributional assumption for the error term. Both classes of AFT models have been popular in the analysis of censored failure time data. The semiparametric AFT model is more flexible and robust to departures from the distributional assumption than its parametric counterpart. However, the semiparametric AFT model is subject to producing biased results for estimating any quantities involving an intercept. Estimating an intercept requires a separate procedure. Moreover, a consistent estimation of the intercept requires stringent conditions. Thus, essential quantities such as mean failure times might not be reliably estimated using semiparametric AFT models, which can be naturally done in the framework of parametric AFT models. Meanwhile, parametric AFT models can be severely impaired by misspecifications. To overcome this, we propose a new type of the AFT model using a nonparametric Gaussian-scale mixture distribution. We also provide feasible algorithms to estimate the parameters and mixing distribution. The finite sample properties of the proposed estimators are investigated via an extensive stimulation study. The proposed estimators are illustrated using a real dataset.     

Expression of hirudin in fed-batch cultures of recombinantSaccharomyces cerevisiae

Summary Fed-batch fermentations were performed to produce hirudin, an anticoagulant protein, from recombinantS. cerevisiae. The structural gene coding for hirudin was combined with theGAL10 promoter for controlled expression and theMFα1 signal sequence for secretion to the growth medium. Control of galactose concentration in a fed-batch mode of operation yielded 110 g/L of final cell density and 260 mg/L of maximum hirudin concentration in the medium, which corresponds to a 3.5-fold increase in cell density and a 4.1-fold enhancement in hirudin concentration compared with the batch fermentation.     

Intracellular pH determination by a 31P-NMR technique. The second dissociation constant of phosphoric acid in a biological system

To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK') value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Hückel equation. Ions (Na+, K+, Mg2+, and Ca2+) and salivary protein affected the pK' value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK' of frog muscle at 24 degrees C.     

The maintenance of free-living amoebae by cryopreservation.

We have successfully cryopreserved free-living amoebae in order to maintain them feasibly under the conditions in our laboratory. The viability of trophozoites was higher when frozen by slow cooling (overall 0.7 degree C/min) than by fast cooling (overall 1.3 degrees C/min). Glycerol and dimethylsulfoxide at the final concentration of 7.5% each was used for cryopreservation of free-living amoebae trophozoites. The survival rate was 2-39% after storage in the liquid nitrogen for 60 days. Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.     

Proton magnetic resonance assay of total and taurine-conjugated bile acids in bile.

Biliary bile acids, coexisting with phospholipid and cholesterol, are partly conjugated with taurine. In the present report we show that total and taurine-conjugated bile acids in bile can be simultaneously and quantitatively measured by high-resolution (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used a 7.05-Tesla NMR spectrometer to obtain the (1)H-NMR spectra of model and biological biles. Only addition of trimethylsilyl-3-propionic acid sodium salt-D(4) (TSP) to each sample as an internal standard was required in preparation for (1)H-NMR measurement. In (1)H-NMR spectra of rat bile, peaks of C-18 methyl protons of bile acids and of C-25 methylene protons on the taurine moiety of taurine-conjugated bile acids were detected at 0.7 ppm and 3.1 ppm, respectively. Peak areas, of C-18 and C-25 peaks, increased in proportion to the concentrations of bile acids or taurine-conjugated bile acids, even in the presence of phospholipid and cholesterol. The accuracy of NMR measurement of total and taurine-conjugated bile acids was confirmed by comparing the results of NMR with those of enzyme-fluorimetry.The results clearly demonstrate that (1)H-NMR spectroscopy can be applied to the quantitative determination of total and taurine-conjugated bile acids in bile without troublesome preparative steps.     

Pyrithione, a zinc ionophore, inhibits NF-kappaB activation.

Pyrrolidine dithiocarbamate (PDTC) suppresses NF-kappaB activity and exhibits cytotoxic effects in bovine cerebral endothelial cells (BCECs), and we have previously reported that these PDTC effects were accompanied by an increase in intracellular zinc levels. To further explore the role of zinc in the modulation of NF-kappaB activation, we studied the effect of pyrithione, a zinc ionophore, on NF-kappaB activation in BCECs. Pyrithione inhibited NF-kappaB activity in a time- and dose-dependent manner. Ca-EDTA, but not Zn-EDTA, prevented pyrithione inhibition of NF-kappaB activity. Pyrithione increased the intracellular zinc level within 15 min. This effect was also abolished by Ca-EDTA, but not by Zn-EDTA. The potency of pyrithione on NF-kappaB inhibition and zinc influx was approximately one order of magnitude more potent than PDTC. These findings establish the regulatory role of intracellular zinc levels on NF-kappaB activity in BCECs.