Appearance of apoptotic cells and granular cells in the silkworm midgut lumen during larval-pupal ecdysis

To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

Triterpenoids from Viburnum suspensum

Three triterpenoids, 3-oxo-11,13(18)-oleanadien-28-oic acid, 24-hydroxy-3-oxo-11,13(18)-oleanadien-28-oic acid, 6β-hydroxy-3-oxo-11,13(18)-oleanadien-28-oic acid have been isolated together with the previously known virgatic acid, vibsanin B and 3-hydroxyvibsanin E from the leaves of Viburnum suspensum. Their structures were determined by spectroscopic methods and by comparison of their NMR spectral data with those of the previously known 11,13(18)-oleanadien-3β-ol.     

New and interesting desmids (Zygnematales, Chlorophyceae) collected from Brazil and Argentina

The morphology and taxonomy of nine desmid taxa belonging to the three genera Closterium, Euastrum and Cosmarium are studied based on freshwater algal collections from Brazil and Argentina. They represent five new varieties (Closterium cynthia De Notaris var. minutum Kanetsuna var. nov., Euastrum attenuatum Wolle var. saitoi Kanetsuna var. nov., Cosmarium laticollum Delponte var. minutum Kanetsuna var. nov., Cosmarium pseudovariolatum Grönblad var. major Kanetsuna var. nov. and Cosmarium taxichondrum Lundell var. yamagishii Kanetsuna var. nov.), two new forms (Euastrum hypochondrum Nordstedt var. hypochondrum f. divergens Kanetsuna f. nov. and Euastrum insulare (Wittrock) Roy var. silesiacum (Gronblad) Krieger f. brasiliense Kanetsuna f. nov.) and one new status (Cosmarium pseudovariolatum Gronblad var. incrassatum (Scott et Gronblad) Kanetsuna f. elonga‐tum (Scott et Gronblad) Kanetsuna stat. nov.). In addition, a new combination (Cosmarium pseudovariolatum Gronblad var. incrassatum (Scott et Gronblad) Kanetsuna comb, nov.) is proposed.     

Protective effect of Bacillus anthracis surface protein EA1 against anthrax in mice

Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA.     

Bending of DNA segments with Saccharomyces cerevisiae autonomously replicating sequence activity, isolated from basidiomycete mitochondrial linear plasmids

Summary Previous studies have indicated that DNA bending is a general structural feature of sequences (ARSs) from cellular DNAs of yeasts and nuclear and mitochondrial genomic DNAs of other eukaryotes that are capable of autonomous replication in Saccharomyces cerevisiae. Here we showed that bending activity is also tightly associated with S. cerevisiae ARS function of segments cloned from mitochondrial linear DNA plasmids of the basidiomycetes Pleurotus ostreatus and Lentinus edodes. Two plasmids, designated pLPO2-like (9.4 kb), and pLPO3 (6.6 kb) were isolated from a strain of P. ostreatus. A 1029 by fragment with high-level ARS activity was cloned from pLPO3 and it contained one ARS consensus sequence (A/T)TTTAT(A/G)TTT(A/T) indispensable for activity and seven dispersed ARS consensus-like (10/11 match) sequences. A discrete bent DNA region was found to lie around 500 by upstream from the ARS consensus sequence (T-rich strand). Removal of the bent DNA region impaired ARS function. DNA bending was also implicated in the ARS function associated with a 1430 by fragment containing three consecutive ARS consensus sequences which had been cloned from the L. edodes plasmid pLLE1 (11.0 kb): the three consecutive ARSs responsible for high-level ARS function occurred in, and immediately adjacent to, a bent DNA region. A clear difference exists between the two plasmid-derived ARS fragments with respect to the distance between the bent DNA region and the ARS consensus sequence(s).     

Accessory carotid body within the parathyroid gland III of the chicken

In the chicken, the cranial and caudal parathyroid glands (parathyroid gland III and IV), which are connected to each other, are located adjacent to the carotid body. In the present study, we found that a mass of glomus cells surrounded by a thick layer of connective tissue was frequently distributed within the parathyroid gland III. The glomus cells in the parathyroid III, as well as those of the carotid body, expressed intense immunoreactivity for serotonin, chromogranin A, and tyrosine hydroxylase but no immunoreactivity for neuropeptide Y. The cells possessed long cytoplasmic processes containing dense-cored vesicles of 70–220 nm in diameter, and were in close association with sustentacular cells. In and around the glomus cell clusters of the parathyroid III, dense networks of varicose fibers showed immunostaining with the monoclonal antibody TuJ1 to a neuronspecific class III -tubulin isotype, c4. Furthermore, the distribution was also detected of numerous galanin-, vasoactive intestinal peptide (VIP)-, substance P-, and calcitonin gene-related peptide (CGRP)-immunoreactive fibers.     

Sex pheromone of the butterbur borer, Ostrinia zaguliaevi

The sex pheromone blend of the butterbur borer, Ostrinia zaguliaevi (Lepidoptera: Pyralidae) was analyzed by means of gas chromatography-electroantennographic detection (GC-EAD), GC-mass spectrometry and a series of wind-tunnel bioassays. Four EAD-active compounds were detected in the female sex pheromone gland extract, and these were identified as tetradecyl acetate (14:OAc), (Z)-9-tetradecenyl acetate (Z9-14:OAc), (E)-11-tetradecenyl acetate (E11-14:OAc) and (Z)-11-tetradecenyl acetate (Z11-14:OAc). The average amounts ± s.d. of the four compounds in a single sex pheromone gland were 7.9±3.7 ng, 10.1±3.2 ng, 1.1±0.5 ng and 11.6±5.1 ng, respectively. In a wind-tunnel bioassay, the ternary blend of Z9-, E11- and Z11-14:OAc at a ratio found in the sex pheromone gland (45:5:50) elicited the same behavioral responses from the males as did virgin females and pheromone gland extract. Removal of any single compound from the ternary blend significantly diminished the pheromonal activity, whereas addition of 14:OAc to the ternary blend had no effect on the males' behavioral responses. Therefore, it was concluded that the sex pheromone blend of O. zaguliaevi is composed of Z9-14:OAc, E11-14:OAc and Z11-14:OAc at a ratio of 45:5:50.     

Sexual dimorphism of the canine roots in theMacaca fuscata

In situ radiographic analysis of the maxillary canines ofMacaca fuscata was conducted on 88 specimens in 44 individuals (23 dry skulls and 21 live animals) in order to examine the number of roots. The left canines were then extracted from ten female skulls for measurement, further radiographic examination, and visual morphological observation. The results showed a clear sexual dimorphism in root morphology: all male canines were clearly distinguished as single-rooted from the radiograph, whereas more than 40% of the female canines were double-rooted. Variation was also found among the single-rooted female canines, in that some of these teeth appeared to have a bifurcated canal. This sexual dimorphism in the number of maxillary canine roots and the individual variation found among the females in root and canal morphology are previously unreported for this species. No observations were attempted on mandibular canines, however, because of the incomplete nature of the sample.     

An artificial maximum neural network: a winner-take-all neuron model forcing the state of the system in a solution domain

A maximum neuron model is proposed in order to force the state of the system to converge to the solution in neural dynamics. The state of the system is always forced in a solution domain. The artificial maximum neural network is used for the module orientation problem and the bipartite subgraph problem. The usefulness of the maximum neural network is empirically demonstrated by simulating randomly generated massive nstances (examples) in both problems. In randomly generated more than one thousand instances our system always converges to the solution within one hundred iteration steps regardless of the problem size. Our simulation results show the effectiveness of our algorithms and support our claim that one class of NP-complete problems may be solvable in a polynomial time.