Studies on Morphological Changes of the Blue-green Alga Nostoc muscorum A with Special Reference to the Role of Light

Morphological changes of Nostoc muscorum A were studied withspecial reference to growth conditions. According to Lazaroff(1973), N. muscorum A has a life cycle dependent on the lightconditions; cells of a coccoid form grow in the dark (aseriatestage) while cells of a filamentous form grow in light (seriatestage). The conversion from the aseriate to the seriate stageis photocontrolled by red stimulation and green suppression.We reexamined (i) the light effect on the morphological formsof N. muscorum A growing under various conditions, includingN₂-fixing and non-fixing, and (ii) the light effect on the controlof the aseriate to seriate conversion in the dark in relationto the effect on the dark growth. Results for (i) indicatedthat cell forms did not necessarily depend on the light butwere determined by growth conditions; under conditions sufficientfor supporting rapid growth, cells grew at the seriate stage,and under insufficient conditions, at the aseriate stage. Thelight effect to induce and support filamentous growth was explainedas the photosynthetic energy supply for improving the growth.Results for (ii) revealed that red light induced the dark aseriateto seriate conversion and at the same time enhanced the darkgrowth after the illumination. Green light suppressed both theconversion and the dark growth stimulated by red light. Thered-green photocontrol of the dark conversion was explainedby the enhancing effect of red light and the inhibitory effectof green light on the dark growth. Morphological changes betweencoccoid and filamentous forms of N. muscorum A are probablynot obligatory for continued growth. ¹Present address: Department of Botany, Faculty of Science,Kyoto University, Kitashirakawa, Kyoto 606, Japan. (Received September 22, 1980; Accepted December 6, 1980)     

Electron donor-specificity observed in photosystem I reactions of membrane fragments of the blue-green alga Anabaena variabilis and the higher plant Spinacea oleracea

Electron donating activities of plastocyanins and c-type cytochromesof various organisms for photosystem I reactions were studiedwith membrane fragments of the blue-green alga Anabaena variabilisand the higher plant Spinacea oleracea. In the Anabaena photosystem I reaction, basic but not acidicplastocyanin and c-type cytochromes acted as efficient electrondonors, while only acidic redox proteins were active in thespinach photosystem I reaction. The selective reactivity ofredox proteins in the two photosystem I reactions was observedwith both plastocyanin (or cytochrome) limited and saturatedconditions. These data support our previous observation that photosystemI of blue-green algae differs from those of other green plantswith respect to specificity to the proteinous electron donor(1). (Received August 17, 1971; )     

Photochemical activity fluorescence and absorption spectrum of the photochemically active chromoprotein (ACP) isolated from the blue-green alga Anabaena cylindrica

Photochemical, spectroscopic and fluorescence characteristicsof the active chromo-protein (ACP) of Anabaena cylindrica werestudied with preparations having different spectroscopic characteristics. Purified ACP preparations occasionally showed spectroscopiccharacteristics differing considerably from those of ordinaryACP. A spectroscopic difference in these preparations was observedonly in the relative absorbance in the 620 nm peak. Absorptionpeaks at 430, 460, 480 and 695 nm remained unchanged. Photochemicalactivity and fluorescence yield at 700 nm were fairly constantin these preparations, when excited at absorption bands otherthan 620 nm. Spectroscopic changes caused by heat and detergent (SDS) treatmentsoccurred only in the relative absorbance of the 620 nm peak.Photochemical and fluorescence characteristics of the treatedACP were the same as those of the ACP originally having a differentabsorption spectrum. A comparison of various ACP preparations indicates that ACPis a complex of two pigments, c-phycocyanin and a pigment witha 695 nm absorption maximum. The reduction in the molecularsize of ACP by SDS-treatment indicates that pigment 695 is farsmaller in its molecular size than c-phycocyanin. A possiblefunction of the two pigments in the photochemical reaction isalso discussed. (Received December 10, 1971; )     

Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis

Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5–9.0 and possessed high thermal stability up to 50°C with 80% of the residual activities compared with the stability seen at the pH range of 6.5–7.0 and temperatures of up to 40°C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass together with cellulases.     

Simple blood‐feeding method for live imaging of gut tube remodeling in regenerating planarians

Live cell imaging is a powerful technique to study cellular dynamics in vivo during animal development and regeneration. However, few live imaging methods have been reported for studying planarian regeneration. Here, we developed a simple method for steady visualization of gut tube remodeling during regeneration of a living freshwater planarian, Dugesia japonica. When planarians were fed blood several times, gut branches were well‐visualized in living intact animals under normal bright‐field illumination. Interestingly, tail fragments derived from these colored planarians enabled successive observation of the processes of the formation of a single anterior gut branch in the prepharyngeal region from the preexisting two posterior gut branches in the same living animals during head regeneration. Furthermore, we combined this method and RNA interference (RNAi) and thereby showed that a D. japonica raf‐related gene (DjrafA) and mek‐related gene (DjmekA) we identified both play a major role in the activation of extracellular signal‐regulated kinase (ERK) signaling during planarian regeneration, as indicated by their RNAi‐induced defects on gut tube remodeling in a time‐saving initial screening using blood‐feeding without immunohistochemical detection of the gut. Thus, this blood‐feeding method is useful for live imaging of gut tube remodeling, and provides an advance for the field of regeneration study in planarians.     

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4‐like viruses isolated in Japan

Bacteriophages (phages) belonging to the family Podoviridae genus N4‐like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4‐like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4‐like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.     

Characterization of inducible nature of MRP3 in rat liver

We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na(+)-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6] transporters. alpha-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment. Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments. Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase. However, the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or alpha-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.     

Proposal of Pseudochattonella verruculosa gen. nov., comb. nov. (Dictyochophyceae) for a formar raphidophycean alga Chattonella verruculosa, based on 18S rDNA phylogeny and ultrastructural characteristics

Chattonella verruculosa Y. Hara et Chihara was re‐examined by molecular methods and microscopic examination. The 18S rDNA phylogenetic analysis clearly indicated that C. verruculosa is a member of the Dictyochophyceae, with a specific affinity to Florenciella parvula. The morphological features in C. verruculosa– namely the proximal helix with two gyres and many scattered DNA‐containing areas in the chloroplasts – display the evolutionary link to the Dictyochophyceae, instead of the Raphidophyceae. Similarly, unique pyrenoid morphologies are shared between C. verruculosa and the dictyochophycean algae. Combining the molecular data and morphological characteristics, C. verruculosa is transferred to Pseudochattonella gen. nov. of the class Dictyochophyceae as Pseudochattonella verruculosa (Y. Hara et Chihara) Hosoi‐Tanabe, Honda, Fukaya, Inagaki et Sako comb. nov.     

A 2-Mb sequence-ready contig map and a novel immunoglobulin superfamily gene IGSF4 in the LOH region of chromosome 11q23.2

Human chromosome 11q23.2 has been proposed to contain a tumor suppressor gene(s) whose deletion has been associated with cancer of the lung and breast and with neuroblastoma. To analyze the genomic structure and to isolate a candidate tumor suppressor gene from this region, we constructed a 2-Mb sequence-ready contig map using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The map comprises a contig of 24 overlapping P1, BAC, and PAC clones. To isolate gene fragments from the region, we performed direct cDNA library screening, exon trapping, EST mapping, and genomic sequencing using the P1, BAC, and PAC clones. Sequence analysis of 5 clones, which spans 23% (458,738 bp) of the region, and extensive gene scanning along the entire region revealed that the region is extraordinarily scarce in genes, but we identified one ubiquitously expressed novel gene and one testis-specific gene fragment. The novel gene, which we call IGSF4 (immunoglobulin superfamily 4), is transcribed into a 1.6- or 4.4-kb RNA encoding a 442-amino-acid protein. It shares strong homology with mouse IGSF-B12 and cell adhesion molecules NCAM1 and NCAM2 within their Ig-like C2-type domains. The IGSF4 gene, a novel gene that is shown to be located in the common loss of heterozygosity region, possesses a number of interesting features and may be good candidate for a tumor suppressor gene.