Chitosan and a fungal elicitor inhibit tracheary element differentiation and promote accumulation of stress lignin-like substance in Zinnia elegans xylogenic culture

We investigated the effect of elicitors on xylem differentiation and lignification using a Zinnia elegans xylogenic culture system. Water-soluble chitosan and a fungal elicitor derived from Botrytis cinerea were used as elicitors. Elicitor addition at the start of culturing inhibited tracheary element (TE) differentiation in a concentration-dependent manner, and 30 μg mL^?1 of chitosan or 16.7 μg mL^?1 of the fungal elicitor strikingly inhibited TE differentiation and lignification. Addition of chitosan (at 50 μg mL^?1) or the fungal elicitor (at 16.7 μg mL^?1) during the culturing period also inhibited TE differentiation without inhibiting cell division, except for immature TEs undergoing secondary wall thickening. Elicitor addition after immature TE appearance also caused the accumulation of an extracellular lignin-like substance. It appears that elicitor addition at the start of culturing inhibits the process by which dedifferentiated cells differentiate into xylem cell precursors. Elicitor addition during culturing also appears to inhibit the transition from xylem cell precursors to immature TEs, and induces xylem cell precursors or xylem parenchyma cells to produce an extracellular stress lignin-like substance.     

蜉蝣目化石的研究进展

回顾了世界蜉蝣目化石研究简史,列出已发表的化石种类名录、分布及年代,共40科、124属、218种,其中包括中国的8属13种;描述了不同地质时期主要蜉蝣化石的分类单元和分布地区.简要介绍了蜉蝣分类系统及系统发育、稚虫食性、与其它昆虫的关系和古生态等方面的研究成果.在本文的第2部分,初步分析了目前该类群所存在的一些问题,并对今后的研究工作进行了展望.     

Chitins and chitosans for the repair of wounded skin,nerve, cartilage and bone

This review provides a balanced integration of the most recent chemical, biochemical and medical information on the unique characteristics of chitins and chitosans in the area of animal/human tissue regeneration. Hemostasis is immediately obtained after application of most of the commercial chitin-based dressings to traumatic and surgical wounds: platelets are activated by chitin with redundant effects and superior performances compared with known hemostatic materials. To promote angiogenesis, necessary to support physiologically ordered tissue formation, the production of the vascular endothelial growth factor is strongly up-regulated in wound healing when macrophages are activated by chitin/chitosan. The inhibition of activation and expression of matrix metalloproteinases in primary human dermal fibroblasts by low MW chitosans prevents or solves problems caused by metalloproteinase-2 such as the hydrolysis of the basement membrane collagen IV. Experimental biocompatible wound dressings derived from chitin are today available in the form of hydrogels, xerogels, powders, composites, films and scaffolds: the latter are easily colonized by human cells in view of the restoration of tissue defects, with the advantage of avoiding retractive scar formation. The growth of nerve tissue has been guided with chitin tubes covalently coated with oligopeptides derived from laminin. The regeneration of cartilage is also feasible because chitosan maintains the correct morphology of chondrocytes and preserves their capacity to synthesize cell-specific extracellular matrix: chitosan scaffolds incorporating growth factors and morphogenetic proteins have been developed. Impressive advances have been made with osteogenic chitosan composites in treating bone defects, particularly with osteoblasts from mesenchymal stem cells in porous hydroxyapatite-chitin matrices. The introduction of azido functions in chitosan has provided photo-sensitive hydrogels that crosslink in a matter of seconds, thus paving the way to cytocompatible hydrogels for surgical use as coatings, scaffolds, drug carriers and implants capable to deliver cells and growth factors. The peculiar biochemical properties of chitins and chitosans remain unmatched by other polysaccharides.     

Chitosan functional properties

Chitosan is a partially deacetylated polymer of N-acetyl glucosamine. It is essentially a natural, water-soluble, derivative of cellulose with unique properties. Chitosan is usually prepared from chitin (2 acetamido-2-deoxy β-1,4-D-glucan) and chitin has been found in a wide range of natural sources (crustaceans, fungi, insects, annelids, molluscs, coelenterata etc.) However chitosan is only manufactured from crustaceans (crab and crayfish) primarily because a large amount of the crustacean exoskeleton is available as a by product of food processing. Squid pens (a waste byproduct of New Zealand squid processing) are a novel, renewable source of chitin and chitosan. Squid pens are currently regarded as waste and so the raw material is relatively cheap. This study was intended to assess the functional properties of squid pen chitosan. Chitosan was extracted from squid pens and assessed for composition, rheology, flocculation, film formation and antimicrobial properties. Crustacean chitosans were also assessed for comparison. Squid chitosan was colourless, had a low ash content and had significantly improved thickening and suspending properties. The flocculation capacity of squid chitosan was low in comparison with the crustacean sourced chitosans. However it should be possible to increase the flocculation capacity of squid pen chitosan by decreasing the degree of acetylation. Films made with squid chitosan were more elastic than crustacean chitosan with improved functional properties. This high quality chitosan could prove particularly suitable for medical/analytical applications. This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of Some Fungal Chitosans

Chitosan-like materials were extracted from five different fungal cells with NaOH and acetic acid, with the yields varying from 1.2 to 10.4% of the dry fungal cell weight. The degree of N-acetylation of the extracts measured by the colloidal titration method varied considerably depending on the individual species. By IR measurements and the Elson-Morgan method, four kinds of the extracts were characterized as chitosan while another one was not.

The degree of N-acetylation and the Cu²⁺ adsorption capacity of the fungal chitosans were measured and compared with those of authentic samples with various degrees of N-acetylation, which were prepared by chemical treatment of authentic chitin and chitosan derived from Crustacea. The Cu²⁺ adsorption capacity of the fungal chitosans was higher than that of the authentic chitosan samples with similar degrees of N-acetylation and independent of the molecular weight of the chitosans from the various sources.     

Reduction of predation risk under the cover of darkness: Avoidance responses of mayfly larvae to a benthic fish

Summary Mayfly larvae of Paraleptophlebia heteronea (McDunnough) had two antipredator responses to a nocturnal fish predator (Rhinichthys cataractae (Valenciennes)): flight into the drift and retreat into interstitial crevices. Drift rates of Paraleptophlebia abruptly increased by 30 fold when fish were actively foraging in the laboratory streams but, even before fish were removed, drift began returning to control levels because larvae settled to the substrate and moved to areas of low risk beneath stones. This drifting response was used as an immediate escape behavior which likely decreases risk of capture from predators which forage actively at night. Surprisingly, drift most often occurred before contact between predator and prey, and we suggest that in darkness this mayfly may use hydrodynamic pressure waves for predator detection, rather than chemical cues, since fish forage in an upstream direction. Although drifting may represent a cost to mayfly larvae in terms of relocation to a new foraging area with unknown food resources, the immediate mortality risk probably out-weighs the importance of staying within a profitable food patch because larvae can survive starvation for at least 2 d. In addition to drifting, mayflies retreated from upper, exposed substrate surfaces to concealed interstitial crevices immediately after a predator encounter, or subsequent to resettlement on the substrate after predator-induced drift. A latency period was associated with this response and mayflies remained in these concealed locations for at least 3 h after dace foraging ceased. Because this mayfly feeds at night and food levels are significantly lower in field refugia under stones, relative to exposed stone surfaces, predator avoidance activity may limit foraging time and, ultimately, reduce the food intake of this stream mayfly.     

Physical characteristics of decolorized chitosan as affected by sun drying during chitosan preparation

Some physical characteristics of decolorized chitosan as affected by sun drying, which was used to replace a bleaching step during chitosan preparation, were evaluated. One bleached and four unbleached chitosans were prepared and dried for 4 h by heat treatment at 60 °C or sun drying. The moisture content of chitosans dried by heat treatment was lower than that of chitosans dried by sun drying. Decoloration of the chitosan could be achieved more effectively by sun drying after deacetylation than by using a bleaching agent in the chitin preparation. Use of a bleaching agent significantly reduced the viscosity of the chitosan solution. A sequence of heat drying and sun drying in chitin and chitosan production (without using a bleaching agent) generally produced a whiter chitosan with higher viscosity without affecting water- and fat-binding capacities, compared to the bleached chitosan.     

Evolutionary divergence in freshwater insects with contrasting dispersal capacity across a sea of desert

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Extraction of chitin and chitosan from housefly,Musca domestica,pupa shells

Chitins and chitosans are some of the most abundant natural polysaccharide materials, and are used to increase innate immune response and disease resistance in humans and animals. In this work, chitin and chitosan from housefly, Musca domestica, pupa shells were obtained by treatment with HCl and NaOH. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve decalcification and deproteinization, respectively. For chitosan extraction, 50% NaOH solution was used to achieve deacetylation. The yields of chitin and chitosan from pupa shells of M. domestica were 8.02% and 5.87%, respectively. The deacetylations of chitosan (from chitin C1 and C2) were 89.76% and 92.39%, respectively, after the first alkali treatment with 50% NaOH (w/w) solution at 105 °C for 3 h and 5 h, respectively. The viscosities of the chitosans (from chitin C1 and C2) were 33.6 and 19.2 cP, respectively.     

Physicochemical characterization of chitin and chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and the yields were 30.0-32.2% with that of chitosan C120 being the highest. The degree of N-deacetylation of chitosans (83.3–93.3%) increased but the average molecular weight (483–526 kDa) decreased with the prolonged reaction time. Crab chitosans showed lower lightness and WI values than purified chitin, chitosans CC and CS but higher than crude chitin. With the prolonged reaction time, the nitrogen (8.9–9.5%), carbon (42.2–45.2%) and hydrogen contents (7.9–8.6%) in chitosans prepared consistently increased whereas N/C ratios remained the same (0.21). Crab chitosans prepared showed a melting endothermic peak at 152.3–159.2 °C. Three chitosans showed similar microfibrillar crystalline structure and two crystalline reflections at 2θ = 8.8–9.0° and 18.9–19.1°. Overall, the characteristics of three crab chitosans were unique and differed from those of chitosan CC and CS as evidenced by the element analysis, differential scanning calorimetry, scanning electron microscopy and X-ray diffraction patterns.     

Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress

The incidence of fluoroquinolone-resistant Shigella strains has risen rapidly, presumably in response to ciprofloxacin antibiotic stress. Understanding the molecular mechanisms underlying this resistance phenotype is critical to developing novel and effective therapeutic strategies. In this study, the frequency of ciprofloxacin-induced mutation was measured in antibiotic resistance genes (gyrA, gyrB, parC, parE, marOR, and marA) of Shigella flexneri. The S. flexneri 2a strain 301 was cultured on Luria–Bertani agar plates containing one of seven different ciprofloxacin concentrations (range: 0.03125–2 μg mL^?1). Resistant colonies were selected for gene-targeted sequencing analysis; the identified point mutations were subsequently confirmed by insertion into antibiotic cassette plasmids and growth under ciprofloxacin stress. The results demonstrated that the seven different ciprofloxacin concentrations produced dose-dependent frequencies of spontaneous mutations: 10^?8 (0.03125 and 0.0625 μg mL^?1), 10^?9 (0.125 μg mL^?1), and <10^?9 (0.25, 0.5, 1, 2 μg mL^?1). PCR sequencing of the ten randomly selected resistant colonies (minimum inhibitory concentrations (MICs) of 0.125 μg mL^?1, n = 5 and 0.25 μg mL^?1, n = 5) revealed that all colonies had mutations in the gyrA gene at either codon 83 (Ser83 → Leu) or 87 (Asp87 → Tyr or → Gly), both of which were confirmed at MIC of 0.125 μg mL^?1. None of the spontaneous mutation colonies exhibited gyrB, parC, parE, marOR, or marA mutations. In conclusion, S. flexneri is normomutable under ciprofloxacin antibiotic stress and fluoroquinolone resistance by spontaneous mutation occurs at a low rate. Codon mutations gyrA 83 and/or gyrA 87 cause a 4-fold increase in the ciprofloxacin MIC, and may represent the natural mechanism of fluoroquinolone resistance.     

Antimicrobial Characteristics of Chitosans against Food Spoilage Microorganisms in Liquid Media and Mayonnaise

Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25°C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.     

TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan

A commercial chitin, regenerated chitin prepared from chitin solutions in 6.8% NaOH and N-acetylated chitosans with degrees of N-acetylation (DNAc) of 77–93% were subjected to oxidization in water with NaClO and catalytic amounts of 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) and NaBr. When regenerated chitin with DNAc of 87% and N-acetylated chitosan with DNAc of 93% were used as starting materials, water-soluble β-1,4-linked poly-N-acetylglucosaminuronic acid (chitouronic acid) Na salts with degrees of polymerization of ca. 300 were obtained quantitatively within 70 min. On the other hand, the original chitin and N-acetylated chitosan with DNAc of 77% did not give water-soluble products, owing to incomplete oxidation. The high crystallinity of the original chitin brought about low reactivity, and the high C2-amino group content of the N-acetylated chitosan with DNAc of 77% led to degradations rather than the selective oxidation at the C6 hydroxyls. The obtained chitouronic acid had low viscosities in water, and clear biodegradability by soil microorganisms.     

Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane

Different molecular weight, 90% deacetylated chitosans were obtained by ultrasonic degradation on 90% deacetylated chitosan at 80 °C for various times.

Ninety percent deacetylated chitosan was prepared from alkali treatment of chitin that was obtained from red shrimp waste. Number average-, viscosity average- molecular weights were measured by gel permeation chromatography and the viscometric method, respectively. Degree of deacetylation was measured by the titration method. Enthalpy, maximum melting temperature, tensile strength and elongation of the membranes, flow rate of permeates and water are properties measured to elucidate the effect of molecular weight of chitosan on the above thermal, mechanical, and permeation properties, respectively of the prepared membranes. Results show tensile strength, tensile elongation, and enthalpy of the membrane prepared from high molecular weight chitosans were higher than those from low molecular weight. However, the permeability show membranes prepared from high molecular weight chitosans are lower than that from those of low molecular weight.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.     

A chitinase with high activity toward partially<Emphasis Type="Italic"> N</Emphasis>-acetylated chitosan from a new,moderately thermophilic,chitin-degrading bacterium,<Emphasis Type="Italic"> Ralstonia</Emphasis> sp. A-471

A moderately thermophilic bacterium, strain A-471, capable of degrading chitin was isolated from a composting system of chitin-containing waste. Analysis of the 16S rDNA sequence revealed that the bacterium belongs to the genus Ralstonia. A thermostable chitinase A (Ra-ChiA) was purified from culture fluid of the bacterium grown in colloidal chitin medium. Purification of the enzyme was achieved mainly by exploiting its binding to the colloidal chitin. The molecular mass of the enzyme was estimated to be 70 kDa and the isoelectric point approximately 4.7. N-terminal amino acid sequencing revealed a sequence of ADPYLKVAYYP, which had high homology (66% identity) with that of chitinase A1 from Bacillus circulans WL-12. The pH and temperature optima were determined to be 5.0 and 70°C, respectively. The enzyme was classified as a retaining glycosyl hydrolase and was most active against partially N-acetylated chitosans. Its activities towards the partially N-acetylated chitosans, i.e. chitosan 7B, chitosan 8B, and chitosan 9B, were about 11-fold, 9-fold, and 5-fold higher than towards colloidal chitin, respectively. Ra-ChiA cleaved (GlcNAc)₆ almost exclusively into (GlcNAc)_2. Activation of Ra-ChiA was observed by the addition of 1 mM Cu²⁺, Mn²⁺, Ca²⁺_, or Mg²⁺. Degradation of the partially N-acetylated chitosan produced oligosaccharides with a degree of polymerization ranging from 1–8; these are products that offer potential application for functional oligosaccharide production.     

Antiviral Action of Methylated β-Lactoglobulin on the Human Influenza Virus A Subtype H3N2

Antiviral activity of methylated β-lactoglobulin (Met-BLG) against H3N2 infected into MDCK cell lines depended on concentration of Met-BLG, viral load, and duration of infection. IC_50% of the hemagglutination activity for 1 and 0.2 MOI (multiplicity of infection) after 24 h of incubation at 37 °C in the presence of 5% CO₂ were 20 ± 0.8 and 17 ± 0.7 μg mL^?1 Met-BLG, respectively. Longer incubation period (4 days) was associated with low IC_50% of the hemagglutination activity (7.1 ± 0.3 μg mL^?1 Met-BLG) and low IC_50% of immuno-fluorescence of viral nucleoproteins (9.7 ± 0.4 μg mL^?1 Met-BLG) when using 0.2 and 0.1 MOI, respectively. A concentration of 25 μg mL^?1 of Met-BLG reduced the amount of replicating virus by about 2 and 1.3 logs when the viral load was 0.01 and 0.1 MOI, respectively, while higher concentrations reduced it by about 5–6 logs. Antiviral action of Met-BLG was coupled with a cellular protective action, which reached 100% when using 0.01 and 0.1 MOI and 83% when using 1.0 MOI. The time of Met-BLG addition after the viral infection was determinant for its antiviral efficacy and for its protection of the infected MDCK cell lines. Anti-hemagglutination action and cell protective action decreased gradually and in parallel with the delay in the time of Met-BLG addition to disappear totally after 10 h delay.     

Consequences of tadpole shrimp predation on mayflies in some Californian ponds

SUMMARY. 1. During two, 1-month studies, the abundance of mayfly nymphs, Callibaetis californicus Banks, in small ponds that contained the tadpole shrimp, Triops longicaudatus (LeConte), often was significantly lower than in ponds without tadpole shrimps. Mayfly abundance was either positively correlated or not significantly associated with that of predacious insects.
2. Larger mayfly nymphs were a greater fraction of the population in ponds with Triops than in ponds without tadpole shrimps. Between 2 and 3 weeks after ponds were flooded, the smallest mayfly nymphs were absent from ponds in which T. longicaudatus densities were >5 individuals per square metre during the summer and >15 individuals per square metre during the autumn.
3. In prey-choice trials, T. longicaudatus consumed predominantly small C. californicus nymphs (mean headwidth ± 1 SD: 0.44 ± 0.05 mm). After encounters with tadpole shrimps, small mayfly nymphs moved relatively shorl distances, were easily overtaken, and were, consequently, more susceptible to tadpole shrimps than larger-sized nymphs.     

Antimicrobial characteristics of chitosans against food spoilage microorganisms in liquid media and mayonnaise.

Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25 degrees C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.     

The influence of predatory fish on mayfly drift: extrapolating from experiments to nature

1. A knowledge of how individual behaviour affects populations in nature is needed to understand many ecologically important processes, such as the dispersal of larval insects in streams. The influence of chemical cues from drift‐feeding fish on the drift dispersal of mayflies has been documented in small experimental channels (i.e. < 3 m), but their influence on dispersal in natural systems (e.g. 30 m stream reaches) is unclear. 2. Using surveys in 10 Rocky Mountain streams in Western Colorado we examined whether the effects of predatory brook trout (Salvelinus fontinalis) on mayfly drift, that were apparent in stream‐side channels, could also be detected in natural streams. 3. In channel experiments, the drift of Baetis bicaudatus (Baetidae) was more responsive to variation in the concentration of chemical cues from brook trout than that of another mayfly, Epeorus deceptivus (Heptageniidae). The rate of brook trout predation on drifting mayflies of both species in a 2‐m long observation tank was higher during the day (60–75%) but still measurable at night (5–10%). Epeorus individuals released into the water column were more vulnerable to trout predation by both day and night than were Baetis larvae treated similarly. 4. Drift of all mayfly taxa in five fishless streams was aperiodic, whereas their drift was nocturnal in five trout streams. The propensity of mayflies to drift was decreased during the day and increased during the night in trout streams compared with fishless streams. In contrast to the channel experiments, fish biomass and density did not alter the nocturnal nature nor magnitude of mayfly drift in natural streams. 5. In combination, these results indicate that mayflies respond to subtle differences in concentration of fish cues in experimental channels. However, temporal and spatial variation in fish cues available to mayflies in natural streams may have obscured our ability to detect responses at larger scales.