Marked variations in patterns of cellulase activity against crystalline- vs. carboxymethyl-cellulose in the digestive systems of diverse, wood-feeding termites

Abstract.  Throughout the history of studies on cellulose digestion in termites, carboxymethyl-cellulose has been preferably used as a substrate for measuring cellulase activity in termites due to its high solubility. However, carboxymethyl-cellulose degradation is not directly related to digestibility of naturally occurring cellulose because many noncellulolytic organisms can also hydrolyse carboxymethyl-cellulose. To address this issue, a comparative study of microcrystalline cellulose digestion is performed in diverse xylophagous termites, using gut homogenates. For those termites harbouring gut flagellates , the majority of crystalline cellulose appears to be digested in the hindgut, both in the supernatant and the pellet. For Nasutitermes takasagoensis , a termite free of gut flagellates, crystalline cellulose is degraded primarily in the midgut supernatant, and partially in the pellet of the hindgut. The fungus-growing termite Odontotermes formosanus , which also does not possess intestinal flagellates, shows only a trace of crystalline cellulose hydrolysis throughout the gut. Comparison of levels of activity against crystalline cellulose with previously reported levels of activity against carboxymethyl-cellulose in the gut of each termite reveals significant differences between these activities. The results suggest that the hindgut flagellates produce commonly cellobiohydrolases in addition to endo-β-1,4-glucanases, which presumably act synergistically to digest cellulose. Preliminary evidence for the involvement of bacteria in the cellulose digestion of N. takasagoensis is also found.     

Extracellular sphingomyelinase induces interleukin-6 synthesis in osteoblasts

In osteoblast-like MC3T3-E1 cells, we have recently reported that sphingosine 1-phosphate among sphingomyelin metabolites acts as a second messenger for tumor necrosis factor-alpha (TNF)-induced interleukin-6 (IL-6) synthesis. In the present study, we investigated the effect of extracellular sphingomyelinase on IL-6 synthesis in MC3T3-E1 cells. Sphingomyelinase stimulated IL-6 synthesis in a time-dependent manner for up to 24 h. This stimulative effect was dose dependent in the range between 1 and 300 mU/ml. Calphostin C, a highly and potent inhibitor of protein kinase C, enhanced sphingomyelinase-induced IL-6 synthesis. DL-Threo-dihydrosphingosine, an inhibitor of sphingosine kinase, significantly inhibited the IL-6 synthesis induced by sphingomyelinase. Sphingomyelinase markedly elicited sphingomyelin hydrolysis. In addition, the effect of a combination of sphingomyelinase and TNF on IL-6 synthesis was synergistic. These results strongly suggest that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblasts, resulting in IL-6 synthesis, and that protein kinase C acts as a negative controller of the IL-6 synthesis.     

The complete mitogenome of the Formosan termite, Coptotermes formosanus Shiraki

The Formosan termite Coptotermes formosanus Shiraki is a well-known invasive pest that causes severe damage to wooden structures in many parts of the world. Although several studies examined its phylogeographic patterns using a few mitochondrial genes, the phylogenetic relationships among C. formosanus are poorly understood because of the small number of mutations known among its mitochondrial genes. To provide a useful genetic tool for further analyses, we analyzed the complete mitochondrial genome sequence of C. formosanus using specimens collected from three isolated islands in the Ryukyu Archipelago of Japan. The circular mitogenome of these termites consisted of genes encoding 22 transfer RNAs, two ribosomal RNAs, and 13 mitochondrial proteins, as is the case for most animal mitochondrial genomes. The G + C content was 34.1%, and the total length varied slightly between 16,234 and 16,236 base pairs. The complete mitochondrial genomes of the three populations were more than 99.9% identical to each other and showed differences at six nucleotide positions. The COII, 12S rRNA, and 16S rRNA genes that are commonly used for phylogenetic analyses revealed only one substitution or no substitutions. The mitogenome sequences determined here should contribute to the design of new molecular markers for the clarification of the historical distribution process of C. formosanus and for further phylogenetic analyses with this and related termite species.     

Attachment structures in Rhizotrochus (Scleractinia): macro‐ to microscopic traits and their evolutionary significance

Marine sessile benthic organisms living on hard substrates have evolved a variety of attachment strategies. Rhizotrochus (Scleractinia, Flabellidae) is a representative azooxanthellate solitary scleractinian coral with a wide geographical distribution and unique attachment structures; it firmly attaches to hard substrates using numerous tube‐like rootlets, which are extended from a corallum wall, whereas most sessile corals are attached by stereome‐reinforced structures at their corallite bases. Detailed morphological and constructional traits of the rootlets themselves, along with their evolutionary significance, have not yet been fully resolved. Growth and developmental processes of spines in Truncatoflabellum and rootlets in Rhizotrochus suggest that these structures are homologous, as they both develop from the growth edges of walls and are formed by transformation of wall structures and their skeletal microstructures possess similar characteristics, such as patterns of rapid accretion and thickening deposits. Taking molecular phylogeny and fossil records of flabellids into consideration, Rhizotrochus evolved from a common free‐living ancestor and invaded hard‐substrate habitats by exploiting rootlets of spines origin, which were adaptive for soft‐substrate environments.     

Structure–activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog’s mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a log P value of 4.0–4.5. On the other hand, an induction test with Epstein–Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.     

Hydrostatic pressure regulates tight junctions, actin cytoskeleton and transcellular ion transport

In the epithelia and endothelia, tight junctions regulate the movement of several substances through the paracellular pathway, maintaining several gradients between apical and basal compartments including osmolality and hydrostatic pressure. In this study, we show that the change of hydrostatic pressure gradient affected tight junctions as well as actin cytoskeleton, cell height and transcellular ion transport. Hydrostatic pressure gradient from basolateral to apical side increased transepithelial conductance and altered claudin-1 localization within several tens of minutes. These changes were promptly restored by the elimination of hydrostatic pressure gradient. Hydrostatic pressure gradient also induced dynamic changes in the actin structure and cell height. We further found that hydrostatic pressure gradient from basolateral to apical side stimulates transcellular Cl⁻ transport. Our present findings indicate that the epithelial cell structures and functions are regulated by the hydrostatic pressure gradient which is generated and maintained by the epithelia themselves.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.     

Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed.     

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.