Chitin in the epidermal cuticle of a vertebrate (Paralipophrys trigloides,Blenniidae, Teleostei)

Lectin binding, endo-chitinase binding and enzymatic degradation studies show that the epidermal cuticle of the bony fishParalipophrys trigloides (Blenniidae) is chitinous. This is the first evidence that a vertebrate species possesses a chitinous tissue. Recently aXenopus gene has been identified which has significant sequence similarity to the catalytic domain of yeast chitin synthase III, a chitin producing enzyme^1,2. Taken together these two findings imply that chitin synthesis capability may be a basic vertebrate feature.     

Biological activity of flucycloxuron,a novel benzoylphenylurea derivative,onTenebrio molitor: comparison with diflubenzuron and triflumuron

Flucycloxuron, a novel benzoylphenylurea (BPU) derivative, exhibited insecticidal activity when injected into newly ecdysed pupae ofTenebrio molitor. Mortality occurs because of defective adult ecdysis. Treatment caused a reduction in both cuticle thickness and incorporation of¹⁴C-labelled precursor into chitin, although it had no significant effect on the protein synthesis. The potencies of other BPU compounds as inhibitors of chitin biosynthesis have been examined and results showed that diflubenzuron was less effective than either flucycloxuron or triflumuron.     

A reinvestigation of the chemistry of the Dufours gland of the formicine ant,Anoplolepis custodiens

The components of individual Dufours glands excised fromAnoplolepis custodiens workers were analysed by GC-MS. In addition to then-alkanes andn-alkenes previously reported² in these glands, primary alcohols (C₁₉-C₂₂), secondary alcohols (C₂₀-C₂₃), 2-ketones (C₂₀-C₂₃) and possibly carboxylate ethyl esters (C₁₉ and C₂₁) were identified as components of these glands. It seems possible that these high-boiling compounds are used by the workers in laying trails on the hot sandy surfaces of their characteristic habitat and in lining of the inner walls of nests, but no standard compounds have been available to us for any behavioral studies.     

Elucidation of the relationship between enzyme activity and internal motion using a lysozyme stabilized by cavity-filling mutations

We investigated the activity and the internal motions of a stabilized mutant hen lysozyme (HEL) in which the residues M12 and L56 were mutated to L and F, respectively (LF mutant HEL). The result of the activity measurements against glycol chitin at various temperatures suggested that the temperature dependence of the activity of LF mutant HEL shifted to the high-temperature side compared with that of wild-type HEL. The detailed internal motions of LF mutant HEL in the absence and presence of a substrate analogue, (NAG)₃, were examined by model-free analysis at 35°C. The results showed that the internal motions of LF mutant HEL in the presence of (NAG)₃ were drastically restricted compared with those in wild-type HEL. Our findings thus suggested that the mutation to the stabilized lysozyme restricted internal motions required for the enzymatic reaction.Received 8 February 2005; accepted 10 March 2005Y. Yoshida and T. Ohkuri contributed equally to this work.     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Instar-specific defensive secretions of stink bugs (Heteroptera: Pentatomidae)

Defensive secretions (allomones) from first-instar nymphs of stink bugs in the subfamily Pentatominae contain (E)-4-oxo-2-decenal as a major constituent, whereas this compound is absent from later instars. In contrast, first instars ofEdessa meditabunda (Edessinae) produce allomones like those of later instars. The C₆ and C₈ (E)-4-oxo-2-alkenals are common, characteristic exocrine compounds of nymphal and adult Heteroptera, but (E)-4-oxo-2-decenal is previously unknown as a major natural product for which a biological role has yet to be established.     

Some citrus chitinases also possess chitosanase activities

Several acidic chitinase and chitosanase isoforms were found in 4-week-old nonembryogenic sweet orange (Valencia [Citrus sinensis (L.) Osbeck]) callus tissue. Two isoforms (designated A1-CF1 and A1-CF2) were purified to homogeneity using HPLC size exclusion, anion exchange, and chromatofocusing techniques. Both hydrolase isoforms exhibited activity with either colloidal chitin or solubilized shrimp shell chitosan. Specific activities for the purified isoforms could not be calculated because of the lack of protein and contamination of ampholytes. However, the specific activities for chitinase and chitosanase after anion exchange were respectively 404 nmol GlcNAc per min per mg protein and 2,475 nmol GlcN per min per mg protein. The M_r for both enzymes was 30,500. The homogeneous proteins cross-reacted in western blots with antiserum against a basic class I potato leaf chitinase.     

Superoxide dismutase activity during the plasmodial life cycle ofPhysarum polycephalum

Summary Superoxide dismutase activity was slow throughout the cell cycle of surface cultures ofPhysarum polycephalum. This activity increased markedly when the organism was induced to spherulate. Glutathione (GSH) and hydrogen peroxide (H₂O₂) concentrations changed very little during the cell cycle. During spherulation GSH decreased; H₂O₂ and the cyanide-resistant respiration of plasmodial homogenates increased.     

Oxidative stress in the moderately halophilic bacteriumDeleya halophila: Effect of NaCl concentration

The sensitivity ofDeleya halophila to oxidative stress caused by hydrogen peroxide (H₂O₂) was found to vary, depending on the NaCl concentration of the growth medium. Pretreatment of the bacteria at a low concentration of H₂O₂ (50 M) protected the cells against the lethal effects of higher levels (1–2 mM) of H₂O₂. Exposure ofD. halophila cells to 50 M H₂O₂ resulted in the induction of several proteins (hydrogen peroxide-inducible proteins, hips). However, the kinetics of induction, the extent of induction and the number of hips appear to be influenced by the salt concentration of the growth medium. Five of the hips exhibited apparent molecular masses identical to those of five heat shock proteins (hsps).     

Tetrasomy and quadruple trisomy in pea

Summary Two off-type plants, morphologically distinguishable from each other and from their respective sister euploid, were isolated in the M₃ generation of pea interchange heterozygotes. Pollen sterility was very high, ranging from 63.0 to 90.0%. Cytologically one of them was tetrasomic (2n+2=16) and the other one was quadruple trisomic (2n+1+1+1+1=18). In the tetrasomic plant 1IV+6II was the most frequent (46.7%) chromosome configuration, while cells with 4III+3II were predominant (40.0% cells) in the quadruple trisomic plant.Financial assistance of C.S.I.R., New Delhi, India is acknowledged.     

Prostaglandin (15S)-PGA2 derivatives in the gorgonianPlexaura homomalla (Esper), formakükenthali Moser

Summary The lipid fraction, 17%, of air-driedPlexaura homomalla formakükenthali, collected in the Caribbean at Puerto Rico, yielded 50% of its weight as the mammalian prostaglandin (15S)-PGA₂ methyl ester. The freeze-dried gorgonian yielded (15S)-PGA₂ largely as the acetate of the methyl ester. (15S)-PGA₂ was also obtained from material collected at St. Croix and at South Caicos. Field observations indicate thatPlexaura kükenthali may be a species separate fromPlexaura homomalla and that it is abundant on some shallow water reefs in the Caribbean. Prostaglandins could not be detected in the lipid fraction of eggs isolated fromPlexaura homomalla (Esper) formahomomalla.Acknowledgments. We gratefully acknowledge support from the Office of Sea Grant, NOAA, Department of Commerce (Grant 047-158-44067), and the use of facilities at the Isla Mayagues Marine Laboratory of the University of Puerto Rico, Mayaguez, the West Indies Laboratory of Fairleigh Dickinson University at St. Croix, U.S.V.I., and the Marine-Biologisch Instituut at Curacao. Special thanks to Dr. F.M. Bayer, Curator, Department of Zoology, National Museum of Natural History-Smithsonian Institution for checking identity of gorgonians used in this study.     

Comparative study of oxalate oxidase in three genotypes ofSorghum vulgare

Summary The presence of an oxalate oxidase (EC 1.2.3.4) has been demonstrated in 15,000×g supernatants prepared from 10-day-old seedlings of three genotypes ofSorghum vulgare: grain sorghum hybrid (CSH-5), grain-cum-forage sorghum (PC-6) and forage sorghum (PC-1). The specific activity of the enzyme in the different tissues of seedlings was found to be present in the order leaves > stems > roots in PC-6 and PC-1, but this order was reversed in CSH-5. A comparison of the different properties of the leaf enzyme of these three genotypes of sorghum revealed that the enzyme has maximum activity in the acidic pH range from 4.0 to 5.0 and in the temperature range from 37°C to 40°C. The enzyme was stimulated by Cu²⁺ and Fe²⁺. The rate of H₂O₂ formation in the enzyme reaction was linear up to 5 min and was stoichiometrically related to oxalate consumption. The enzyme is unaffected by Na⁺ at physiological concentration (0.15 M). The superiority of this enzyme over moss and other plant enzymes for enzymic determination of urinary oxalate is discussed.     

Effect of xanthurenic acid on P-450-dependent biotransformation by molting glands in vitro

Incubation of molting glands from the crayfishProcambarus clarkii (Y-organ) and the silkwormBombyx mori (prothoracic gland) with 23,24-[²H₄]-2-deoxyecdysone resulted in the production of deutero-ecdysone; this biotransformation was inhibited in the presence of xanthurenic acid. When the experiments were performed under an¹⁸O₂ atmosphere, the¹⁸O atom was introduced into ecdysone, as confirmed by mass spectrometry. We therefore suggest that xanthurenic acid inhibits P-450-dependent hydroxylation of 2-deoxyecdysone. However, deutero-2-deoxyecdysone was not converted to 3-dehydroecdysone when using Y-organs in vitro, although it is a major product. We therefore conclude that the biosynthetic pathway of ecdysteroids inP. clarkii branches at an early step.     

Hyper-tetraploids in pea

Summary Cytomorphologically, five hypertetraploids; three hexasomic (4n+2=30), one double pentasomic (4n+1+1=30) and one multiple aneuploid tetraploid (4n+2+1+1+1=33), where isolated in the C₃ generation of pea autotetraploids. Plants with 30 chromosomes were morphologically very distinct from their euploids and characterized by variation in plant height and morphology of leaves. Hexasomic and double pentasomic tetraploids were characterized by the presence of a hexavalent and two pentavalents, respectively. The multiple aneuploid tetraploid showed very vigorous growth and varying frequencies of hexavalents and pentavalents. All the five aneuploids showed high Anaphase-I anomalies; pollen sterility ranged from 50% to 81%.Financial assistance of C.S.I.R., New Delhi, India is acknowledged.     

Role of <Emphasis Type="Italic">N</Emphasis>-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation

Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc₃Man₉GlcNAc₂ glycans after trimming with endoplasmic reticulum (ER) -glucosidases and -mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc₁Man₉GlcNAc₂) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man₈GlcNAc₂ isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc₂ along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man₅GlcNAc₁ by the action of cytosolic endo--N-acetylglucosaminidase and -mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc₃Man₉GlcNAc₂-P-P-Dol, and the unfolded protein response, which enhances synthesis of components of the quality control system.Received 26 January 2004; accepted 25 February 2004     

Polyoxin fungicides: Demonstration of insecticidal activity due to inhibition of chitin synthesis

Summary Polyoxin A, a selective inhibitor of chitin synthetase, was found to be insecticidal when injected into the abdomen of grasshoppers nymphs, with an LD₅₀ of 1.26±0.20 g per insect. Symptomatology and the absence of any toxicity toward adult insects indicate that toxicity is due to interference with cuticle deposition.Acknowledgment. I thank Henry Bork for his expert technical assistance.     

Influence of phosphorus supply and light intensity on mycorrhizal response inPisum-Rhizobium-Glomus symbiosis

The influence of mycorrhizal colonization withGlomus mosseae on parameters of N₂ fixation and plant growth was studied in pot experiments with pea plants (Pisum sativum L.) infected withRhizobium leguminosarum and supplied with varied levels of phosphorus (P) and nitrogen (N). Reduced light intensities were used to evaluate the dependence of the microsymbionts on assimilate supply. In plants grown with low P supply, mycorrhization increased the concentration of P in shoots, and thus N₂ fixation. Reduced light intensity significantly depressed mycorrhizal colonization and nodule growth in low-P plants. When P supply did not limit plant growth and N₂ fixation, however, the percentage of mycorrhizal colonization was reduced due to the higher P status, and the microsymbionts were not impaired by low light intensities. To maximize carbohydrate supply, another experiment was carried out at high light intensity of 900 mol m^–2s^–1 and with non-limiting P supply. Nitrogen fertilization, given as starter N, enhanced plant growth, but delayed nodule formation. Towards flowering, nodulation rapidly increased, but less so inGlomus inoculated plants. After 28 days mycorrhizal plants were lower in shoot dry weight, nodule dry weight and nitrogenase activity. The results suggest that under many, but not all, environmental conditions the host plant is able to restrict mycorrhizal colonization and, thus, to prevent impairment ofRhizobium symbiosis.deceased in May 1994     

Cycles of juvenile hormone esterase activity during the juvenile hormone-driven cycles of oxygen consumption in pupal diapause of flesh flies

Summary During diapause O₂ consumption in fly pupae is a cyclic event (4-day periodicity at 25°C) driven by cycles of juvenile hormone activity. Levels of juvenile hormone esterase activity change systematically during the cycle, with highest activity observed at the nadir of the O₂ consumption cycle.Supported in part by grant 88-37153-3473 from the USDA Competitive Grants Office and grant 23-088 from the USDA Forest Service. Thanks to Dr Ming Tu Chang for her helpful advice.     

Adipokinetic hormones: cell and molecular biology

Adipokinetic hormones AKH I (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH₂) and AKH II (pGlu-Leu-Asn-Phe-Ser-Trp-Gly-Thr-NH₂) are synthesized by neurosecretory cells (NSC) of the corpora cardiaca (CC) in the locust,Schistocerca gregaria. These NSC constitute a homogeneous peptide factory as each cell synthesizes both AKH I and AKH II. This homogeneity makes the CC an excellent system in which to study aspects of neuropeptide biosynthesis. This report summarizes recent findings on AKH inactivation and metabolism, as well as on AKH prohormone processing and biosynthesis.     

RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in Candida albicans

The calcineurin pathway has been reported to be essential for the development of azole resistance in Candida albicans. The depletion or ectopic over-expression of RTA2 increased or decreased susceptibility of C. albicans to azoles, respectively. CaCl₂- induced activation of the calcineurin pathway in wildtype C. albicans promoted resistance to azoles, while the Ca ²⁺ chelator (EGTA), calcineurin inhibitors (FK506 and cyclosporin A) and the deletion of RTA2 blocked the resistance-promoting effects of CaCl₂. Furthermore, we found that RTA2 was up-regulated in a calcineurin-dependent manner. The depletion of RTA2 also made the cell membrane of C. albicans liable to be destroyed by azoles and RTA2 over-expression attenuated the destroying effects. Finally, the disruption of RTA2 caused an increased accumulation of dihydrosphingosine (DHS), one of the two sphingolipid long-chain bases, by decreasing release of DHS. In conclusion, our findings suggest that RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 14 July 2008; received after revision 29 August 2008; accepted 16 September 2008