Occurrence and Some Properties of Cellulase in the Filtrates of Conidiospores and Mycelia of Pyricularia oryzae Cavara

Occurrence of cellulase activity was demonstrated in the filtrates of germinating conidiospores and growing mycelia of P. oryzae. Activity and some properties of cellulase in the filtrate of mycelia grown on rice plant powder as carbon source were compared among various strains.

Cellulase activity (C₁ and C_x enzymes; cellulose and carboxymethylcellulose as substrates, respectively) in the filtrate of germinating conidiospores was detected in the pathogenic T–l (Ken 53–33) strain as well as nonpathogenic 0 (THU 3 × 1) strain of P. oryzae. The activity was higher in the former than the latter strains. Cellulase activity (C_x enzyme) in the filtrate of growing mycelia was detected in the four strains used, T–l (Ken 53–33), C–3 (N 87), N–1 (H373), and 0 (THU 3 × 1). Cellulase activity (C_x enzyme) in the filtrate of mycelia was optimal at pH 5.0 and 40°C, and stable up to 40°C. Their properties did not differ significantly except for the pH-activity curve at alkaline side among various strains; but cellulase activity (C₁ enzyme) was found to be correlated with their pathogenicity except for the case of C–3 strain.     

High-performance liquid chromatographic method for determination of DX-9065a, a novel anticoagulant, in human urine and feces using cation-exchange solid-phase extraction

A simple HPLC method for determination of DX-9065a in human urine and feces was developed. The drug was extracted by Bond Elut CBA, a cation-exchange solid-phase extraction cartridge. The extracted drug was analyzed by HPLC with UV detection at 242 nm. With this extraction procedure, no interfering peaks were observed. The method developed was validated and showed adequate precision and accuracy. This method was applied to human clinical samples obtained from healthy Japanese volunteers who had orally received the drug. Using this method, the excretion profile of the drug in human after oral administration was revealed for the first time.     

Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II).

Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile. This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G)via a protonated Schiff base. Various mutants were engineered to have a single cysteine in the F-helix. In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate. The fluorescence of the isolated protein increased with increasing illumination. These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule. This reaction occurred only at the M-state and not at the O-state. The implications are discussed.     

Detection of lactate dehydrogenase B4 in human hemolysate of patients deficient in lactate dehydrogenase B subunit activity using enzyme immunoassay

We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)- B₄ with antiacetylated LDH-B₄ Fab-horse-radish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B₄ concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B₄ concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.This work was supported in part by grants in aid for Scientific Research from the Ministry of Education, Japan (59570998), and from the Clinical Pathology Research Foundation of Japan.     

Structural polymorphism of murine factor B controlled by a locus closely linked to the H-2 complex and demonstration of multiple alleles

New alleles of murine factor B (Bf) protein were demonstrated. When ethylenediaminetetraacetic acid (EDTA)-plasmas from inbred and wild mice were analyzed by isoelectro-focusing (IEF) and immunofixation, murine Bf proteins were visualized as distinct protein bands in all mice tested. Four variants of murine Bf could be demonstrated in a large number of tested mice: Bf 1 (isoelectro-focusing point (P.I.) range of 5.8–6.1) exemplified by B10 and B10.BR, Bf 2 (P.I. range of 5.8–6.0) exemplified by B10.MOL (OHM), Bf 3 (P.I. range of 5.6–5.9) exemplified by B10.MOL (TEN2) and Mus musculus (Mus m.) subspecies Chc, Bf4 (P.I. range of 6.0–6.3) exemplified by Mus m. subspecies Shh. The genetic linkage between S locus and Bf locus was studied with two backcross progenies — [B 10.BR × (B10.BR × Mus m. subspecies Chc)F₁] and [B 10.BR × (B10.BR × Mus m. subspecies Shh)F₁]. Totally, 256 backcross progenies were typed for Bf type and for Ss type (plasma level of the fourth complement protein regulated by S locus). The results indicated that murine Bf was controlled by a single codominant locus located close to the H-2 complex because no mouse showing recombination between Bf locus and S locus was found.     

Genotypic analysis of families with lactate dehydrogenase A(M) deficiency by selective DNA amplification

Summary Genomic DNA prepared from LDH-A-deficient whole blood was amplified by the polymerase chain reaction technique using two primers specific for the active human LDH-A gene. The amplified fragment was examined by direct agarose gel electrophoresis, and a deletion of 20 base pairs (bp) in exon 6 of the LDH-A gene was found. The results permitted a clear distinction between the homozygous mutant, the heterozygous mutant, and wild-type genotypes. Moreover, HinfI digestion and direct sequencing of the amplified product confirmed the results from direct agarose gel electrophoresis. Four families, including 18 individuals, were shown to contain the same mutation, that is a 20-bp deletion in exon 6. All genotypes were consistent with their biochemical phenotypes as evaluated by the ratio of LDH-B to LDH-A subunits in erythrocytes. Thus, all four known affected families in Japan have been shown to carry the same mutant gene, which may have been derived from a single mutational event.     

The fine structure of the proliferative cells of the mouse intestine as revealed by electron microscopic autoradiography with 3H-thymidine

Summary The duodenal and colonic epithelia in mice were observed with electron microscopic autoradiography 2, 5 and 24 hours after a single injection of ³H-thymidine. After 2 hours, in the duodenum, silver grains are found in many undifferentiated cells, in a few young goblet cells, in some crystal-containing cells, and in some lymphocytes. In the colon after 2 hours silver grains are seen in some undifferentiated cells, and in many young goblet cells. Undifferentiated cells are characterized by a few short microvilli, poorly developed rough-surfaced endoplasmic reticulum, abundant free ribosomes, and a few apical moderately dense granules. In normal animals, absorptive cells seem to arise from undifferentiated cells, and goblet cells — from younger goblet cells. Undifferentiated cells could also become young goblet cells. Crystal-containing cells, which may not be of epithelial origin, proliferate in the epithelium in the adult animal.     

Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release: relationship with the Gag-to-GagPol ratio

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.     

Gripping ease in southern green stink bugs Nezara viridula L. (Heteroptera: Pentatomidae): Coping with geometry,orientation and surface wettability of substrate

The southern green stink bug Nezara viridula L. (Heteroptera, Pentatomidae) is highly polyphagous, preferring apically situated seeds and fruits on more than 150 plant species belonging to over 30 plant families all over the world. This forces them to move over highly variable terrains, including plant stems, leaves, pods and buds, which requires efficient attachment. Stink bugs have long slender legs and feet (tarsi) equipped with paired curved claws, paired soft adhesive pads (pulvilli), and flattened lanceolate hairs (setae), which arise ventrally on the first and second foot segments (tarsomeres). To characterize their attachment abilities on well‐defined test substrates, here we comparatively measured and analyzed the traction forces of bugs walking horizontally and vertically on hydrophilic (water attractive) and hydrophobic (water repellent) glass plates and rods. The latter correspond to the geometry of preferred feeding sites of stink bugs in the field. The results show a clear contribution of tarsal flattened lanceolate hairs to the stink bug's attachment. Higher traction forces are generated on a glass rod than on a glass plate, corresponding to up to individual maximum of 43 times the stink bug's body weight. Substrate hydrophobicity promotes the attachment, while the measured forces are up to eight times lower when tarsal hairs are disabled. The combination of smooth and hairy tarsal pads results in a remarkable attachment ability, which enables N. viridula to climb unstable apical plant parts, and supports their invasive behavior and global dispersion.     

Expression of the Dan gene during chicken embryonic development.

The Dan gene was first identified as the putative rat tumor suppressor gene and encodes a protein structurally related to Cerberus and Gremlin in vertebrates. Xenopus DAN, as with Cerberus and Gremlin, was demonstrated to block bone morphogenetic protein (BMP) signaling by binding BMPs, and to be capable of inducing additional anterior structures by ectopic overexpression in Xenopus embryos. DAN, thus, is suggested to play pivotal roles in early patterning and subsequent organ development, as in the case of other BMP antagonists. In this report, we isolated the chicken counterpart of Dan. Chicken Dan is mainly expressed in the cephalic and somitic mesoderm and several placodes during organ development.