Ikuo Yamaoka

Site of secretion and properties of endogenous endo-β-1,4-glucanase components from Reticulitermes speratus (Kolbe), a Japanese subterranean termite

Two endo-beta-1,4-glucanase components (YEG1 and YEG2) of the endogenous cellulase from the Japanese subterranean termite, Reticulitermes speratus, were purified to homogeneity using gel filtration and hydroxylapatite chromatography, and their enzymatic properties were investigated. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), YEG1 and YEG2 had M(r) of 42 kDa and 41 kDa, respectively. Both components had an optimal pH of 6.0, an optimal temperature of 50 degrees C and were stable at 40 degrees C for at least 30 min. Both components showed high activity on sodium carboxymethylcellulose (CMC), 73.6 U/mg protein for YEG1 and 83.4 U/mg protein for YEG2. The K(m) values of YEG1 and YEG2 on CMC were 1.83 mg/ml and 1.48 mg/ml, respectively. YEG1 did not hydrolyse cellotetraose or cellotriose, whereas YEG2 hydrolysed cellotetraose to cellobiose and cellotriose to cellobiose and glucose. Both YEG1 and YEG2 hydrolysed cellopentaose to cellotriose and cellobiose. Neither component hydrolysed cellobiose. The hydrolytic products from crystalline cellulose (Sigmacell type 20) by YEG1 and YEG2 were cellobiose and a trace amount of glucose. Polyclonal mouse anti-serum raised against YEG2 crossreacted with YEG1, suggesting a common origin for both components. Using this anti-serum, Western blotting and immunohistochemistry showed the presence of YEG1 and YEG2 in the salivary glands, but not in the midgut epithelium. The data suggest that the salivary glands are the site of secretion of endo-beta-1,4-glucanase in R. speratus.

Symbiotic Associations with Protists

Progress in understanding the symbiosis between protists and termites has not matched that between prokaryotes and termites. Methods are now available for the isolation of pure cultures of trichomonads and hypermastigids, although only a few have been cultivated. Sufficient molecular data are now available to construct tentative phylogenetic trees. Molecular data indicate that these organisms are amongst the most primitive eukaryotes lacking mitochondria; the trichomonads also use a prokaryote-like 70S ribosome. In metabolic terms, more is known about the cellulolytic protists but only in outline.

Localization of symbiotic clostridia in the mixed segment of the termite Nasutitermes takasagoensis (Shiraki).

ABSTRACT Phylogeny and the distribution of symbiotic bacteria in the mixed segment of the wood-eating termite Nasutitermes takasagoensis (Shiraki) were studied. Bacterial 16S rRNA genes (rDNA) were amplified from the mixed segment of the gut by PCR, and two kinds of sequences were identified. The phylogenetic tree was constructed by neighbor-joining and maximum parsimony methods to identify symbionts harbored in the mixed segment. They are classified as low-G+C-content gram-positive bacteria and are most closely related to the genus Clostridium. The distribution of these bacteria throughout the whole gut was examined by PCR using specific primers, which suggested that they are confined to the mixed segment despite the presence of bacteria throughout the gut. In situ hybridization indicated that the symbiotic bacteria were localized to the ectoperitrophic space between the midgut wall and the peritrophic membrane in the mixed segment. Electron microscopy revealed the close association between these bacteria and the mesenteric epithelium, suggesting that they have some interactions with the gut tissue of termites.

aproctous, a locus that is necessary for the development of the proctodeum in Drosophila embryos, encodes a homolog of the vertebrate Brachyury gene

The proctodeum of the Drosophila embryo originates from the posterior end of the blastoderm and forms the hindgut. By enhancer-trap mutagenesis, using a P-element-lacZ vector, we identified a mutation that caused degeneration of the proctodeum during shortening of the germ band and named it aproctous (apro). Expression of the lacZ reporter gene, which was assumed to represent expression of the apro gene, began at the cellular blastoderm stage in a ring that encompassed about 10–15% of the eggs length (EL) and included the future proctodeum, anal pads, and posterior-most part of the visceral mesoderm. In later stages, strong expression of lacZ was detected in the developing hindgut and anal pads. Expression continued in the anal pads and epithelium of the hindgut of larvae; the epithelium of the hindgut of the adult fly also expressed lacZ. The spatial patterns of the expression of lacZ in various mutants suggested that the embryonic expression of apro was regulated predominantly by two gap genes, tailless (tll) and huckebein (hkb): tll is necessary for the activation of apro, while hkb suppressed the expression of apro in the region posterior to 10% EL. Cloning and sequencing of the apro cDNA revealed that apro was identical to the T-related gene (Trg) that is a Drosophila homolog of the vertebrate Brachyury gene. apro appears to play a key role in the development of tissues derived from the proctodeum.

Novel tissue units of regional differentiation in the gut epithelium of Drosopbila, as revealed by P-element-mediated detection of enhancer

We analysed spatial patterns of expression of a lacZ reporter gene in the gut of Drosophila larvae that had been transformed with a P-element-lacZ vector to identify regional differences in gene expression. lacZ-positive epithelial cells formed distinct domains with discrete transverse and longitudinal boundaries along the gut tube. Boundaries were often found at sites at which morphological boundaries were not obvious. The gut epithelium was subdivided into 36 compartments by the boundaries. We refer to these novel compartments as “tissue compartments”. The lacZ-positive domain of each strain appeared as a single tissue compartment or as a combination of several tissue compartments. The tissue compartment is considered to be a unit of regional differentiation. The spatial organization of the tissue compartments may represent the “floor plan”, determined by genes that control the regional differentiation of this nonsegmental organ.

Morphology of the Digestive System in the Wood-Feeding Termite Nasutitermes takasagoensis (Shiraki) (Isoptera: Termitidae)

Abstract The morphologies of epithelial cells throughout the alimentary canal of the wood-feeding termite Nasutitermes takasagoensis (Shiraki) were examined. The digestive tract consists of four principal portions, which are the foregut, the midgut, the mixed segment and the hindgut. The midgut epithelium is primarily composed of columnar cells and degenerative cells. Most columnar cells have one or more autophagic vacuoles at cell apexes, suggesting a rapid turnover of the midgut cells. In the mixed segment, the mesenteric epithelium occupies half of the gut wall and the proctodeal epithelium covers the remaining wall. Extensive invaginations of the basal membrane are characteristic of the mesenteric columnar cells, suggesting active transport of an ionic fluid. The hindgut can be divided into five segments, the first of which is a simple tube lined with a thick cuticle, termed the first proctodeal segment. The epithelium of the third segment, the paunch, consists of cuboidal cells, which are covered by multiple cuticular layers. The apical membrane of these epithelial cells forms regular invaginations, suggesting that they have an absorptive function. In the anterior paunch, numerous spirochetes are found adhered to the gut wall. Our observations indicate that termites such as N. takasagoensis appear to have developed structures that enable more efficient interactions with intestinal microorganisms, particularly by the elongation and differentiation of the hindgut and the creation of the mixed segment.

Ultrastructure of the developing fibrocartilage of the Os penis of rat

ABSTRACT

Cytochemical and ultrastructural studies of the scales of the Amoeba Cochliopodium bilimbosum (Testacea)

Summary Cochliopodium bilimbosum, a testacean amoeba, has characteristic scales on its dorsal surface. The structural components of these scales and the process of scale formation were examined by electron microscopy and also by cytochemical staining for polysaccharides. Abundant vesicles and vacuoles containing rudimentary scale-like structures were observed in the cytoplasm. During the transport of vacuoles from Golgi area to the dorsal surface of the organism, the scales became fully developed. Fine grains of the cytochemical reaction products were detected in the scales, and the intensity of staining increased during the transport of the vacuoles. These results suggest that the scale components are assembled in vesicles that are derived from the Golgi complex. The vesicles then fuse to form vacuoles and, during transport of the vacuoles to the dorsal surface, the organization of the scales may be completed both morphologically and biochemically.

Close Correlation between the Distribution of Tenascin and That of Actin Filaments in the Mouse Urethral Mesenchyme during Active Morphogenesis

The distribution of tenascin, an extracellular matrix glycoprotein, and that of actin filaments were studied in the developing urethra of mouse embryos by antitenascin immunofluorescent and rhodamine-phalloidin staining. Tenascin appeared transiently in the urethral mesenchyme at the site of active morphogenesis in which the urethral epithelium separated from the surface epithelia of the glans and prepuce, being tubular, and the bilateral mesenchymes lining the preexisting urethral epithelium were seamed together in the ventral side of the tubular urethra immediately after the epithelial separation. The spatially and temporally restricted distribution of tenascin corresponded well to that of mesenchymal cells which possessed many actin filaments. These observations suggest that tenascin is involved in the cytoskeletal organization of mesenchymal cells in the active phase of morphogenesis.

SYMBIOSIS IN TERMITES

Termites are divided into two types, the lower termite, which possesses symbiotic protozoa and several species of bacteria in the intestine and the higher termite, which has no protozoa but several species of symbiotic bacteria in the intestine. The digestive system for cellulose differs in each. In the higher termites, the digestive system is complex, since several problems remain unresolved. In the lower termites, cellulose digestion depends on the intestinal protozoa and symbiotic relationships between the termite and its intestinal protozoa, first published by Cleveland in 1924 (xcCleveland, 1924A). Recent reports, however, show that the symbiotic relationship in the digestive system involving cellulose is more complex. xcYamaoka and Nagatani (1975) reported that the termites themselves produce a cellulase which differs from the cellulase produced by the intestinal protozoa. Since the concept of the cellulose digestion system has changed significantly, the role of intestinal bacteria and their diversity in termites are presented here.