Chitin synthesis in Spodoptera frugiperda wing imaginal discs. II. Selective action of chlorfluazuron on wheat germ agglutinin binding and cuticle ultrastructure

The action of the chitin synthesis inhibitor, chlorfluazuron, was investigated in Spodoptera frugiperda wing imaginal discs cultured in vitro. Electron microscopy and cytochemical labeling with a lectin, wheat germ agglutinin (WGA), were used to monitor morphogenesis, as well as the presence and localization of chitin and non-polymerized N-acetyl-D-glucosamine (GlcNAc). Chlorfluazuron (CFA) selectively inhibited 20- hydroxyecdysone–stimulated chitin synthesis and procuticle deposition in imaginal discs, without otherwise affecting their morphogenesis. Tracheole migration, evagination, exocytosis, and endocytosis in the epithelial cells, and the presence of non-polymerized GlcNAc in the extracellular matrix, were observed in both CFA-treated and control wing discs. On the other hand, CFA prevented the appearance of WGA-labeled chitin in newly formed procuticle, while the deposition of proteinaceous cuticulin and epicuticle was unaffected. A brief treatment with CFA resulted in WGA labeling of non-polymerized GlcNAc, but not chitin in the procuticle region. The lack of chitin in CFA treated wing discs was correlated with the appearance of an amorphous, non-lamellar procuticle region. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Mechanistic insights into replication termination as revealed by investigations of the Reb1-Ter3 complex of Schizosaccharomyces pombe

Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the “sweepase” Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.     

Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development

Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration–dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2–3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.     

Post-translational control of de novo cell wall formation during Blastocladiella emersonii zoospore germination: feedback regulation of hexosamine biosynthesis.

The Blastocladiella emersonii zoospore does not contain sufficient total hexosamine to account for the chitin content of the cell wall formed during germination. It is not deficient in the enzymes needed to synthesize chitin from fructose-6-phosphate and glutamine. The enzymes of hexosamine biosynthesis are located differently in the zoospore than chitin synthetase. Uridine-5′-diphospho-N-acetylglucosamine (UDPGlcNAc), the end product of hexosamine synthesis and a substrate for chitin synthesis, reversibly inhibits the activity of only the first pathway-specific enzyme at concentrations below that estimated to exist in the zoospore. UDPGlcNAc combines with the enzyme-glutamine complex in direct competition with fructose-6-phosphate. Uridine nucleoside phosphates, produced through the utilization of UDPGlcNAc in chitin synthesis, directly compete with the inhibitory effects of UDPGlcNAc, while other nucleoside phosphates can enhance the inhibition due to UDPGlcNAc. The data are consistent with the simultaneous binding of UDPGlcNAc at two enzyme sites to inhibit catalysis — the substrate (fructose-6-phosphate) site and the uridine nucleoside phosphate site. The hexosamine pathway can be negatively regulated, as it is in the zoospore, by UDPGlcNAc and can be positively regulated, as it is during zoospore germination, by lowering UDPGlcNAc concentration and raising UDP + UTP concentrations. Other variations in these metabolites could regulate hexosamine biosynthesis during other phases of the B. emersonii life cycle.     

Some Effects of Diflubenzuron on Growth and Sporogenesis in Streptomyces spp.

Diflubenzuron, an insect growth regulator that blocks chitin deposition in insect cuticles, was tested for its effects on morphogenesis of Streptomyces spp. Use of diflubenzuron resulted in reduced dominance of spore hairs, reduced the width of the outer wall, and prevented formation of the inner spore wall in S. bambergiensis. In S. coelicolor, diflubenzuron altered the structure of the fibrillar pattern of spore envelopes. Exposure to diflubenzuron resulted in small increases in exported protein and in a ca. 20% increase in chitinase in both Streptomyces spp.     

Characterization of chitin and chitin synthase from the cellulosic cell wall fungusSaprolegnia monoi¨ca

The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (K_i 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.     

Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum

Chitin synthases catalyze the formation of β-(1,4)-glycosidic bonds between N-acetylglucosamine residues to form the unbranched polysaccharide chitin, which is the major component of cell walls in most filamentous fungi. Several studies have shown that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of the genus Aspergillus although little research on this topic has been done in Penicillium chrysogenum. We used BLAST to find the genes encoding chitin synthases in P. chrysogenum related to chitin synthase genes in Aspergillus nidulans. Three homologous sequences coding for a class III chitin synthase CHS4 and two hypothetical proteins in P. chrysogenum were found. The gene which product showed the highest identity and encoded the class III chitin synthase CHS4 was studied in detail. To investigate the role of CHS4 in P. chrysogenum morphogenesis, we developed an RNA interference system to silence the class III chitin synthase gene chs4. After transformation, mutants exhibited a slow growth rate and shorter and more branched hyphae, which were distinct from those of the original strain. The results also showed that the conidiation efficiency of all transformants was reduced sharply and indicated that chs4 is essential in conidia development. The morphologies of all transformants and the original strain in penicillin production were investigated by light microscopy, which showed that changes in chs4 expression led to a completely different morphology during fermentation and eventually caused distinct penicillin yields, especially in the transformants PcRNAi1-17 and PcRNAi2-1 where penicillin production rose by 27 % and 41 %, respectively.     

A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the “neck filaments” that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.     

Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress

In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.     

Endochitinase Is Transported to the Extracellular Milieu by the eps-Encoded General Secretory Pathway of Vibrio cholerae

The chiA gene of Vibrio cholerae encodes a polypeptide which degrades chitin, a homopolymer of N-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans. chiA has a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa. A 52-bp region with promoter activity was found immediately upstream of the chiA open reading frame. Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity by V. cholerae required chiA. Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for β,1-4 glycosidic bonds located between GlcNAc monomers in chitin. Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein. Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes of V. cholerae and by the closely related bacterium Aeromonas hydrophila. Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by the eps system. The eps system is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA. These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential for eps-dependent secretion.Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is a major component of the cell walls of fungi and the integuments of crustaceans and insects (38). The molecule is one of the most abundant biopolymers in nature and is used by many microorganisms as a source of carbon. Utilization of chitin as a nutrient usually requires degradation of the molecule to GlcNAc monomers. Complete degradation of chitin in both prokaryotes and eukaryotes is a two-step process which involves successive hydrolysis of the β,1-4 glycosidic bonds linking the GlcNAc subunits. In the first stage, endochitinase binds and degrades tetrameric and longer polymeric forms of GlcNAc to produce the disaccharide chitobiose. In the second step, chitobiase hydrolyzes chitobiose to GlcNAc monomers. The enzymes for chitin degradation are usually coordinately regulated and in several organisms are induced by chitosan, chitobiose, GlcNAc, or glucosamine (2, 7, 44).Members of the family Vibrionaceae thrive in marine environments where chitin is abundant. It is not surprising that many Vibrionaceae evolved systems for utilizing chitin as a nutrient source. Chitinases have been identified in Vibrio vulnificus (56, 61), V. harveyi (57), and V. parahemolyticus (29, 30). Nucleotide sequence analysis indicated that the chitinase of V. harveyi is homologous with human hexosamindase, indicating that the two enzymes, as well as other chitinases, are members of a phylogenically related group (56).V. cholerae is a human intestinal pathogen that resides in brackish and marine waters. In vitro experiments established that V. cholerae has the potential to use chitin as a sole source of carbon for growth (15). It is likely, therefore, that production of chitinase (29, 30, 42) by V. cholerae provides the bacterium with a readily available nutrient source in aquatic environments. Hydrolysis of chitin by V. cholerae is an extracellular process that requires expression of epsE, one of a cluster of genes in the eps locus (43, 46–48). Several proteins of V. cholerae are dependent on the eps system for extracellular transport, including cholera toxin (CT), an undefined protease, and a chitinase activity (43, 48). Although expression of chitinase activity has been reported for V. cholerae, the enzyme responsible for the activity has not been identified. To further characterize the extracellular chitinase of V. cholerae, we cloned a gene encoding chitinase activity. Here we report the nucleotide sequence of a cloned endochitinase gene and establish that the protein encoded by that gene is secreted to the extracellular environment by an eps-dependent mechanism.     

Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

We examined the mechanism of attachment of the marine bacterium Vibrio harveyi to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of V. harveyi to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of V. harveyi to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of V. harveyi to chitin.     

Anti-Inflammatory Effects of Rebamipide Eyedrop Administration on Ocular Lesions in a Murine Model of Primary Sj?gren's Syndrome

Background

Topical therapy is effective for dry eye, and its prolonged effects should help in maintaining the quality of life of patients with dry eye. We previously reported that the oral administration of rebamipide (Reb), a mucosal protective agent, had a potent therapeutic effect on autoimmune lesions in a murine model of Sjögren''s syndrome (SS). However, the effects of topical treatment with Reb eyedrops on the ocular lesions in the murine model of SS are unknown.

Methods and Finding

Reb eyedrops were administered to the murine model of SS aged 4–8 weeks four times daily. Inflammatory lesions of the extraorbital and intraorbital lacrimal glands and Harderian gland tissues were histologically evaluated. The direct effects of Reb on the lacrimal glands were analyzed using cultured lacrimal gland cells. Tear secretions of Reb-treated mice were significantly increased compared with those of untreated mice. In addition to the therapeutic effect of Reb treatment on keratoconjunctivitis, severe inflammatory lesions of intraorbital lacrimal gland tissues in this model of SS were resolved. The mRNA expression levels of IL-10 and mucin 5Ac in conjunctival tissues from Reb-treated mice was significantly increased compared with those of control mice. Moreover, lactoferrin production from lacrimal gland cells was restored by Reb treatment.

Conclusion

Topical Reb administration had an anti-inflammatory effect on the ocular autoimmune lesions in the murine model of SS and a protective effect on the ocular surfaces.     

Impact of nitrogen supply on growth,steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni

This work investigated the agronomic, physiological and biochemical response of Stevia rebaudiana Bertoni grown under different nitrogen (N) rates. A pot trial in open air conditions was set up in 2012 with the aim to evaluate the effect of four N rates on the biometric and productive characteristics, steviol glycoside (SG) content as well as on leaf gas exchanges, chlorophyll fluorescence, photosynthetic pigments, Rubisco activity and N use efficiency. N deficiency caused a decrease in leaf N content, chlorophylls and photosynthetic CO₂ assimilation, resulting in a lower dry matter accumulation as well as in reduced SG production. The application of 150 kg N ha^? 1 seems to be the most effective treatment to improve rebaudioside A (Reb A) content, Reb A/stevioside ratio, photosynthetic CO₂ assimilation, stomatal conductance, N use efficiency, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) and PSII efficiency. The results demonstrate that by using an appropriate N rate it is possible to modulate the SG biosynthesis, with a significant increase in the Reb A content and in the ratio between Reb A and stevioside. This finding is of great relevance in order to obtain a raw material designed to meet consumer needs and bio-industry requirements for high-quality, Reb A content, and safe and environmentally friendly products.     

Armigeres subalbatus prophenoloxidase III: Cloning,characterization and potential role in morphogenesis

It has long been suggested that phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning and melanotic encapsulation of pathogens. Here we report that a mosquito PO, designated Armigeres subalbatus prophenoloxidase III (As-pro-PO III), is likely involved in the morphogenesis in mosquito. Expression profile analysis found that As-pro-PO III mRNA is persistently expressed in adult mosquitoes and is not significantly affected by blood feeding, microfilariae inoculation, or Escherichia coli inoculation, but expression levels of As-pro-PO III fluctuated in larval and pupal stages. Knockdown of As-pro-PO III expression in pupae using double-stranded RNA resulted in high pupal mortality and deformed adults that subsequently died following emergence. Promoter activity analyses by electrophoretic mobility-shift assays and transfection assays suggest that the As-pro-PO III gene is positively regulated by a putative Zeste motif, a developmental regulatory element. These results suggest that As-pro-PO III is associated with morphogenesis of mosquitoes.     

Phenotypic characterization of a trifluoperazine-resistant mutant ofMucor rouxii

A trifluoperazine-resistant (TFP¹) mutant (strain G5) ofMucor rouxii was isolated and some biochemical and physiological parameters were studied. It resisted up to 250 μM TFP compared to 100 μM observed for the wild-type strain. At this drug concentration the mutant strain G5 germinated, grew, exhibited yeast-mycelium transition, and chitin synthesisin vivo. The mutant strain presentedin vitro levels of calmodulin activity similar to those of the wild-type, but with less sensitivity to inhibition by TFP. Also, with regard to spore germination and cell growth, mutant G5 presented cross-resistance to calmidazolium, another potent anticalmodulin drug. Partially purified chitin synthetase preparations of mutant G5 exhibited a diminished enzymatic activity, compared to the wild-type. The results presented in this work suggest the participation of a Ca²⁺-calmodulin complex in growth and differentiative processes ofMucor and substantiate the role of this complex in chitin synthesis.