Conformational analysis of chitobiose and chitosan

The relaxed potential energy surfaces of chitobiose were calculated based on the MM3-force field by optimizing dimer structures on a 10° grid spacing of the torsional angles about the glycosidic bonds (Φ,Ψ). The 36 conformations; the four combinations of the hydroxymethyl group orientations coupled with the nine of the secondary group ones— were assumed for each Φ,Ψ conformation. The four conformations, each differing in the hydroxymethyl group orientations, were considered for the whole Φ,Ψ space, and all the 36 conformations, for the restricted space of low energy. While the resulting energy map and the structures of the energy minima were similar to those proposed for cellobiose in many respects, more restricted energy profile was suggested for the relaxed map of chitobiose where differences in the energy level between the global minimum and the local minima were within 5.4 kcal/mol, compared with the equivalent value of 3.6 kcal/mol for cellobiose. Further depression of the global minimum occurred when the acidic residue was used. The Monte Carlo samples of the chitosan chain were generated based on the relaxed map to predict the unperturbed coil dimension in solution. The chitosan chains showed Gaussian behavior at x = 500 (x, degree of polymerization) and gave the characteristic ratio C_x, of about 70, which was much larger than the experimental values observed for the chitosan and cellulosic chains. © 1994 John Wiley & Sons, Inc.     

Role of pyrenoids in the CO2-concentrating mechanism: comparative morphology, physiology and molecular phylogenetic analysis of closely related strains of Chlamydomonas and Chloromonas (Volvocales)

The morphology of the pyrenoid and the physiology of the CO₂-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969, Cd. macrostellata Lund SAG 72.81, Cd. bipapillata Bourrelly SAG 11-47, and Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are closely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. augustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, electron-dense pyrenoid matrix surrounded by starch granules was present, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of Cr. insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher concentration of Rubisco molecules than the thylakoid region (thylakoid and stroma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chlamydomonas strains examined in this study and that of Cd. reinhardtii Dangeard. In all six strains examined, the presence of the CCM was indicated by relatively high photosynthetic affinities for CO₂ (low values of K_0.5(CO₂)). However, differences in the inorganic carbon (Ci) pools were recognized in relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cd. radiata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2–3 similar to the two pyrenoid-less, CCM-containing strains of Chloromonas previously examined (E. Morita et al., 1998, Planta 204: 269–276). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups. Received: 21 August 1998 / Accepted: 30 November 1998     

TAXONOMIC STUDY OF TWO NEW GENERA OF FUSIFORM GREEN FLAGELLATES,TABRIS GEN. NOV. AND HAMAKKO GEN. NOV. (VOLVOCALES,CHLOROPHYCEAE)1

On the basis of LM, we isolated strains of two species of fusiform green flagellates that could be assigned to former Chlorogonium (Cg.) Ehrenb. One species, “Cg.”heimii Bourr., lacked a pyrenoid in its vegetative cells and required organic compounds for growth. The other was similar to Cg. elongatum (P. A. Dang.) Francé and “Cg.”acus Nayal, but with slightly smaller vegetative cells. Their molecular phylogeny was also studied based on combined 18S rRNA, RUBISCO LSU (rbcL), and P700 chl a‐apoprotein A2 (psaB) gene sequences. Both species were separated from Chlorogonium emend., Gungnir Nakada and Rusalka Nakada, which were formerly assigned to Chlorogonium. They were accordingly assigned to new genera, Tabris Nakada gen. nov. and Hamakko (Hk.) Nakada gen. nov. as T. heimii (Bourr.) Nakada comb. nov. and Hk. caudatus Nakada sp. nov., respectively. Tabris is differentiated from other genera of fusiform green flagellates by its vegetative cells, which only have two apical contractile vacuoles and lack a pyrenoid in the chloroplast. Hamakko, on the other hand, is distinguishable by the fact that its pyrenoids in vegetative cells are penetrated by flattened thylakoid lamellae.     

Inhibition and redistribution of NHE3, the apical Na+/H+ exchanger, by Clostridium difficile toxin B

NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.     

A new scenario of plastid evolution: plastid primary endosymbiosis before the divergence of the “Plantae,” emended

A recent hypothesis on the origin of eukaryotic phototrophs proposes that red algae, green plants (land plants plus green algae), and glaucophytes constitute the primary photosynthetic eukaryotes, whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis, whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary endosymbiotic events involving a phototrophic eukaryote and a host cell. However, the phylogenetic relationships among the three lineages of primary photosynthetic eukaryotes remained unresolved because previous nuclear multigene phylogenies used incomplete red algal gene sequences derived mainly from Porphyra (Rhodophyceae, one of the two lineages of the Rhodophyta), and lacked sequences from the Cyanidiophyceae (the other red algal lineage). Recently, the complete nuclear genome sequences from the red alga Cyanidioschyzon merolae 10D of the Cyanidiophyceae were determined. Using this genomic information, nuclear multigene phylogenetic analyses of various lineages of mitochondrion-containing eukaryotes were conducted. Since bacterial and amitochondrial eukaryotic genes present serious problems to eukaryotic phylogenies, basal eukaryotes were deduced based on the paralogous comparison of the concatenated - and -tubulin. The comparison demonstrated that cellular slime molds (Amoebozoa) represent the most basal position within the mitochondrion-containing organisms. With the cellular slime molds as the outgroup, phylogenetic analyses based on a 1,525-amino acid sequence of four concatenated nuclear genes [actin, elongation factor-1( EF-1), -tubulin, and -tubulin] resolved the presence of two large, robust monophyletic groups and the basal eukaryotic lineages (Amoebozoa). One of the two groups corresponded to the Opisthokonta (Metazoa and Fungi), whereas the other included various lineages containing primary and secondary plastids (red algae, green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, dinoflagellates, and Heterolobosea, for which the red algae represented the most basal lineage. Therefore, the plastid primary endosymbiosis likely occurred once in the common ancestor of the latter group, and the primary plastids were subsequently lost in the ancestor(s) of organisms within the group that now lacks primary plastids. A new concept of Plantae was proposed for phototrophic and nonphototrophic organisms belonging to this group on the basis of their common history of plastid primary endosymbiosis. This new scenario of plastid evolution is discussed here, and is compared with recent genome information and findings on the secondary endosymbiosis of the Euglena plastid.     

Water extract of the root of Lindera strychnifolia slows down the progression of diabetic nephropathy in db/db mice

We examined a possible preventive effect of Linderae radix (LR), the root of Lindera strychnifolia, on the progression of diabetic nephropathy. Water extract of Linderae radix (LR extract) was orally administered to the C57BL/KsJ-db/db (db/db) mice, a model of genetic diabetes, at a dose of 730 mg/kg/day for 12 week. The LR extract treatment did not affect glucose metabolism and systolic pressure. However, it resulted in a better renal function as evaluated by creatinine clearance (Ccr) and serum creatinine than the control; Ccr and serum creatinine were progressively worsened in controls (0.13+/-0.01 (l/day) and 0.69+/-0.04 (mg/dl), respectively) whereas unchanged in the treated group (0.24+/-0.03 (l/day), p<0.05 and 0.53+/-0.04 (mg/dl), p<0.05, respectively). Kidneys of the LR extract-treated group showed glomeruli with greater area and cell population, smaller glomerular sclerotic index, and less fibrosis in glomeruli, where apoptotic rate of glomerular cells were decreased compared with the control kidneys. Furthermore, renal TGF-beta(1) expression was decreased in the LR extract-treated group. These findings suggest that the LR therapy can be a novel therapeutic approach against diabetic nephropathy.     

The Simplest Integrated Multicellular Organism Unveiled

Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.     

l-Glutamic Acid Fermentation

It is well known that biotin has a marked effect on l-glutamic acid fermentation.

The authors have intended to find strains which are independent of the amounts of biotin in the culture medium. As a result, oleic acid-requiring mutants were obtained from a strain of Brevibacterium thiogenitalis which is an auxotroph for biotin. The growth of the mutant was remarkably stimulated by Tween 20, 40, 60, Ca ions and a small amount of corn steep liquor. And also, the mutant was found to have lost its requirement for biotin and showed growth response only to oleic acid or unsaturated fatty acids.

The effect of biotin, oleic acid and other unsaturated fatty acids on the production of l-glutamic acid was investigated by using an oleic acid-requiring mutant of Brevibacterium thiogenitalis No. 653. The results described in the present paper showed that the oleic acid-requiring mutant D-248 produced a large amount of l-glutamic acid in the excess biotin-contaming media, and that oleic acid seemed to be completely replaced by other unsaturated fatty acids such as palmitoleic acid and linoleic acid.