β-structure from the microfibillar proteins of Merino wool

The β-structure of S-caboxymethyl derivatives of microfibrillar proteins isolated from Merino wool was investigated by X-ray diffraction for comparison with the structur of β-keratin. The S-carboxymethylated microfibrillar proteins(SCMKA) w well-oriented β-films of SCMKA weer obtained by stretching the SCMKA cast films in steam up to about 300% extesnsion. It was found that the reflections in β-pattern of SCMKA may be indexed on a pseudo-orthorhombic unit cell with a =0.94 nm, b = 0.66 nm and c = nm, where the ab, and c axes are in the direction of the interchain hydrogen bonding, the main chain(fibre axis) and the side chain, respectively. The unit cell dimesnions evaluated for SCMKA were almost the same as those for β-keratin, suggeting that few peptide sequences containing S-carboxymethyl cystine may be involved in the formation of β-structure from SCMKA.     

Molecular phylogeny of Punctaria mageshimensis reveals evidence for its transfer to Spatoglossum as S. mageshimense (Dictyotales,Phaeophyceae)

Taxonomy of the little‐studied brown algal species Punctaria mageshimensis (Ectocarpales s.l.) was reexamined by molecular phylogeny and morphology. In the genetic analyses of newly collected specimens using plastid rbcL and psaA gene sequences, the specimens morphologically referable to P. mageshimensis were phylogenetically distant from Ectocarpales s.l. and were included in the clade of Spatoglossum (Dictyotales). Morphological reexamination of the type specimen and newly collected specimens confirmed its systematic position in Dictyotales: Branched thallus; cushion‐shaped rhizoidal holdfast occasionally forming secondary holdfast at the bottom of the thallus; many discoidal plastids without pyrenoid per cell; tetrasporangium‐like reproductive structures with dark, homogeneous cell content; occurrence of hair tufts. Genetically P. mageshimensis was most related to a reported sequence of Spatoglossum asperum, but P. mageshimensis was considerably different from S. asperum as well as other known Spatoglossum species in the deep habitat and in having scarcely‐branched lanceolate and considerably thickened thallus. In conclusion, we propose the transfer of P. mageshimensis to Spatoglossum as S. mageshimense comb. nov.     

Tinocladia sanrikuensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

The new species Tinocladia sanrikuensis sp. nov. H.Kawai, K.Takeuchi & T.Hanyuda (Ectocarpales s.l., Phaeophyceae) is described from the Pacific coast of the Tohoku region, northern Japan based on morphology and DNA sequences. The species is a spring–summer annual growing on lower intertidal to upper subtidal rocks and cobbles on relatively protected sites. T. sanrikuensis has a slimy, cylindrical, multiaxial erect thallus, slightly hollow when fully developed, branching once to twice, and resembles T. crassa in gross morphology. The erect thalli are composed of a dense medullary layer, long subcortical filaments, and assimilatory filaments of 11–35 cells, up to 425 μm long and curved in the upper portion. Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most closely related to T. crassa and has the same basic thallus structures but differs in having thinner and longer assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes support the distinctness of this species.     

Distribution and variable expression of secretory pathway protein reticulocalbin in normal human organs and non-neoplastic pathological conditions.

Reticulocalbin (RCN) is one member of the Ca(2+)-binding proteins in the secretory pathway and is localized in the endoplasmic reticulum. RCN may play a role in the normal behavior and life of cells, although its detailed role remains unknown. Overexpression of RCN may also play a role in tumorigenesis, tumor invasion, and drug resistance. The new antibody for human RCN is used in the distribution of RCN in normal human organs of fetuses and adults with or without inflammation. Immunohistochemical examination demonstrated a broad distribution of RCN in various organs of fetuses and adults, predominantly in the endocrine and exocrine organs. However, RCN expression was heterogeneous in each constituent cell of some organs. Among non-epithelial organs, vascular endothelial cells, testicular germ cells, neurons, and follicular dendritic cells showed strong staining. Plasma cells were the only RCN-positive cells among hematopoietic and lymphoid cells. In inflammatory conditions, RCN expression was enhanced in both epithelial and non-epithelial cells. Heterogeneous expression of RCN indicates that the amount of RCN needed for cell behavior and life may be variable, depending on each cell type and, therefore, RCN may be helpful in establishing the cell origin of neoplasms in some organs. However, further study is needed to establish the significance of RCN in tumorigenesis and in some peculiar features of neoplasms.     

Plus and minus sexual agglutinins from Chlamydomonas reinhardtii

Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic alpha-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head-head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft-shaft interactions mediated by carbohydrates displayed in polyproline II configurations.     

Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells

Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-beta) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-beta does.     

Dual Biosynthesis Pathway for Longer-Chain Polyamines in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N⁴-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N⁴-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N¹-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k_cat/K_m value for N¹-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N¹-aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k_cat/K_m value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N¹-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N¹-aminopropylagmatine. Furthermore, spermine and N⁴-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.Polyamines are positively charged aliphatic compounds. Putrescine [4], spermidine [34], and spermine [343] are common polyamines observed in various living organisms, from viruses to humans (16). Polyamines, which play important roles in cell proliferation and cell differentiation (19, 34), are thought to contribute to adaptation against various stresses (9, 26). In thermophilic microorganisms, polyamines contribute to growth under high-temperature conditions. Indeed, in the thermophilic bacterium Thermus thermophilus, a mutant strain lacking the enzyme related to polyamine biosynthesis shows defective growth at high temperatures (23). Furthermore, thermophilic archaea and bacteria possess long-chain and branched-chain polyamines such as N⁴-aminopropylspermidine [3(3)4], N⁴-aminopropylspermine [3(3)43], and N⁴-bis(aminopropyl)spermidine [3(3)(3)4], in addition to common polyamines (11, 13, 14). N⁴-aminopropylspermine was detected in the cells of thermophiles, such as Saccharococcus thermophilus, thermophilic Bacillus and Geobacillus spp. (Bacillus caldolyticus, B. caldotenax, B. smithii, Geobacillus stearothermophilus, and G. thermocatenulatus), Caldicellulosiruptor spp. (C. kristjanssonii and C. owensensis) and Calditerricola spp. (C. satsumensis and C. yamamurae) (10, 12, 22), but it was not detected in archaea. These unique polyamines are thought to support the growth of thermophilic microorganisms under high-temperature conditions. An in vitro study indicated that long-chain and branched-chain polyamines effectively stabilized DNA and RNA, respectively (32).Polyamines are synthesized from amino acids such as arginine, ornithine, and methionine (26). In most eukaryotes, putrescine is synthesized directly from ornithine by ornithine decarboxylase (34). Plants and some bacteria possess additional or alternative putrescine biosynthesis pathways in which putrescine is synthesized from arginine via agmatine (18, 31, 35). In this pathway, agmatine is synthesized by arginine decarboxylase, and agmatine is converted to putrescine by agmatine ureohydrolase or a combination of agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase. Longer polyamines are then produced by the addition of the aminopropyl group from decarboxylated S-adenosylmethionine. This pathway is shown on the left in Fig. ​Fig.11 (pathway I). On the other hand, the thermophilic bacterium T. thermophilus possesses a unique polyamine-biosynthetic pathway (23) in which spermidine is synthesized from agmatine via N¹-aminopropylagmatine by aminopropyl transferase followed by ureohydrolase, as shown on the right in Fig. ​Fig.11 (pathway II).Open in a separate windowFIG. 1.Predicted biosynthetic pathway of polyamines in T. kodakarensis. (A) Predicted biosynthetic pathway. Pyruvoyl-dependent arginine decarboxylase proenzyme (TK0149), arginine/agmatine ureohydrolases (TK0240/TK0474/TK0882), aminopropyl transferase (TK0147), and pyruvoyl-dependent S-adenosylmethionine decarboxylase proenzyme (TK1592) are shown based on the genome analysis. (B) Structures of unique polyamines.A sulfur-reducing hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, was isolated from Kodakara Island, Kagoshima, Japan (1, 21). This archaeon grows at temperatures between 60°C and 100°C but optimally at 85°C. Under low- or high-temperature-stressed conditions, T. kodakarensis produces cold- or heat-inducible chaperones to adapt to unfavorable growth environments (4, 5, 30). The lipid composition of the membrane also changes depending on the growth shift (20). In addition to acting as such tolerance factors, polyamines have been suggested to play an important role in maintaining nucleosomes in high-temperature environments (15). A complete genome analysis of T. kodakarensis has been performed, and the pathway of polyamine biosynthesis has been predicted (Fig. ​(Fig.1)1) (6, 7). It has been speculated that putrescine is synthesized from arginine via agmatine by arginine decarboxylase (PdaD_Tk) and agmatine ureohydrolase. Long- and/or branched-chain polyamines are then produced by the addition of the aminopropyl group derived from decarboxylated S-adenosylmethionine. Previously, we revealed that PdaD_Tk catalyzed the first step of polyamine biosynthesis and was essential for cell growth (6). The strain DAD, which lacks the gene pdaD_Tk, does not grow in medium without agmatine. Archaeal cells are known to use agmatine to synthesize agmatidine, which is an agmatine-conjugated cytidine found at the anticodon wobble position of archaeal tRNA^Ile (17). Agmatine is important for agmatidine synthesis as well as long-chain polyamine. In the present study, we focused on the subsequent steps in polyamine biosynthesis, especially from agmatine to spermidine. T. kodakarensis possesses three agmatine ureohydrolase homologues (TK0240, TK0474, and TK0882); however, it is unclear which one is dominantly functional in T. kodakarensis cells. In a closely related genus, Pyrococcus, TK0474 and TK0882 orthologues have been identified, but the TK0240 orthologue is missing in Pyrococcus genomes. In Pyrococcus horikoshii, PH0083, which is an orthologue of TK0882, was shown to possess agmatine ureohydrolase activity (8). TK0882, hence, appears to possess agmatine ureohydrolase activity as well. It is unclear whether other agmatine ureohydrolase homologues (TK0240 and TK0474) are involved in polyamine synthesis and cell growth in T. kodakarensis. In addition to agmatine ureohydrolase, aminopropyl transferase plays a crucial role in the synthesis of polyamines. TK0147 was annotated first as spermidine synthase and shares sequence identity with aminopropyl transferase (PF0127) from Pyrococcus furiosus (3). It is therefore expected to harbor the function of aminopropyl transferase for long-chain-polyamine synthesis. Recombinant PF0127 showed broad amine acceptor specificity for agmatine, 1,3-diaminopropane (3), putrescine, cadaverine (5), sym-nor-spermidine (33), and spermidine. While maximal catalytic activity was observed with cadaverine, agmatine was most often preferred on the basis of the k_cat/K_m value (3), suggesting that pathway II is a dominant route for polyamine synthesis in P. furiosus. In the present study, various disruptants lacking genes for polyamine biosynthesis were constructed in order to understand the physiological roles of these enzymes in T. kodakarensis. The cell growth profiles and cytoplasmic polyamines of the wild type and the disruptants were analyzed and compared. Recombinant enzymes were also purified and characterized. The obtained results are expected to provide useful information regarding the specific roles of polyamines in thermophiles.