The internal cuticle of Cirsium horridulum (Asteraceae) leaves

Leaf internal cuticle has not previously been studied in detail, and yet its existence has profound implications for the path of water movement. The internal cuticle forms a uniform layer on the inner periclinal epidermal walls that border substomatal cavities. This cuticle is continuous with the external cuticle through the stomatal pores. The thickness of the internal cuticle on nonstomatal epidermal cells is approximately one-third that of the external cuticle on the same cells. On both the abaxial and adaxial sides of the leaf the internal cuticle forms irregularly shaped islands bordered by mesophyll cells. The size of the islands coincides with the epidermal area of the substomatal cavity. The internal cuticle remains intact and connected to the external cuticle after incubation in cellulytic enzymes. After treatment with sulfuric acid or chloroform, both cuticles remain intact. The autofluorescence of both cuticles is increased by staining with auramine O. These results indicate that large portions of the leaf epidermis are covered by both an internal and an external cuticle.     

Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.     

Development and ultrastructure of the rigid dorsal and flexible ventral cuticles of the elytron of the red flour beetle,Tribolium castaneum

Insect exoskeletons are composed of the cuticle, a biomaterial primarily formed from the linear and relatively rigid polysaccharide, chitin, and structural proteins. This extracellular material serves both as a skin and skeleton, protecting insects from environmental stresses and mechanical damage. Despite its rather limited compositional palette, cuticles in different anatomical regions or developmental stages exhibit remarkably diverse physicochemical and mechanical properties because of differences in chemical composition, molecular interactions and morphological architecture of the various layers and sublayers throughout the cuticle including the envelope, epicuticle and procuticle (exocuticle and endocuticle). Even though the ultrastructure of the arthropod cuticle has been studied rather extensively, its temporal developmental pattern, in particular, the synchronous development of the functional layers in different cuticles during a molt, is not well understood. The beetle elytron, which is a highly modified and sclerotized forewing, offers excellent advantages for such a study because it can be easily isolated at precise time points during development. In this study, we describe the morphogenesis of the dorsal and ventral cuticles of the elytron of the red flour beetle, Tribolium castaneum, during the period from the 0 d-old pupa to the 9 d-old adult. The deposition of exocuticle and mesocuticle is substantially different in the two cuticles. The dorsal cuticle is four-fold thicker than the ventral. Unlike the ventral cuticle, the dorsal contains a thicker exocuticle consisting of a large number of horizontal laminae and vertical pore canals with pore canal fibers and rib-like veins and bristles as well as a mesocuticle, lying right above the enodcuticle. The degree of sclerotization appears to be much greater in the dorsal cuticle. All of these differences result in a relatively thick and tanned rigid dorsal cuticle and a much thinner and less pigmented membrane-like ventral cuticle.     

Modeling the bacterial flagellum by an elastic network of rigid bodies

Bacteria such as Escherichia coli propel themselves by rotating a bundle of helical filaments, each driven by a rotary motor embedded in the cell membrane. Each filament is an assembly of thousands of copies of the protein flagellin which assumes two different states. We model the filament by an elastic network of rigid bodies that form bonds with one another according to a scheme suggested by Namba and Vondervistz (1997 Q. Rev. Biophys. 30 1-65) and add additional binding sites at the inner part of the rigid body. Our model reproduces the helical parameters of the 12 possible polymorphic configurations very well. We demonstrate that its energetical ground state corresponds to the normal helical form, usually observed in nature, only when inner and outer binding sites of the rigid body have a large axial displacement. This finding correlates directly to the elongated shape of the flagellin molecule. An Ising Hamiltonian in our model directly addresses the two states of the flagellin protein. It contains an external field that represents external parameters which allow us to alter the ground state of the filament.     

The effects of rigid motions on elastic network model force constants

Elastic network models provide an efficient way to quickly calculate protein global dynamics from experimentally determined structures. The model's single parameter, its force constant, determines the physical extent of equilibrium fluctuations. The values of force constants can be calculated by fitting to experimental data, but the results depend on the type of experimental data used. Here, we investigate the differences between calculated values of force constants and data from NMR and X-ray structures. We find that X-ray B factors carry the signature of rigid-body motions, to the extent that B factors can be almost entirely accounted for by rigid motions alone. When fitting to more refined anisotropic temperature factors, the contributions of rigid motions are significantly reduced, indicating that the large contribution of rigid motions to B factors is a result of over-fitting. No correlation is found between force constants fit to NMR data and those fit to X-ray data, possibly due to the inability of NMR data to accurately capture protein dynamics.     

The stabilisation of insect cuticles

The possible means by which insect cuticle is stabilised are examined: evidence is presented that secondary interactions between the components are very important in stabilisation. Cuticular proteins are shown to be able to adopt stable secondary structures. It is concluded that cuticle may well be stabilised by changes in the conformation of the proteins, changes in the secondary bonding between the proteins and the inclusion of a polymer filler. Crosslinking of the cuticular components may be of little consequence: the stabilisation would be a result of cuticular dehydration.     

Gas permeability of plant cuticles

Cuticles from the adaxial surface of Citrus aurantium L. leaves and from the pericarp of Lycopersicon esculentum L. and Capsicum annuum L. were isolated enzymatically and their oxygen permeability was determined. Isolated cuticles were mounted between a gaseous and an aqueous compartment with the physiological outer side of the membrane facing the gaseous compartment. Permeability for oxygen was characterized by permeability (P) and diffusion (D) coefficients. P and D were independent of the driving force (gradient of oxygen concentration) across the cuticle, thus, Henry's law was obeyed. P values for the diffusion of oxygen varied between 3·10^-7 (Citrus), 1.4·10^-6 (Capsicum), and 1.1·10^-6 (Lycopersicon) m·s^-1. Extraction of soluble lipids from the cuticles increased the permeability. By treating the cutin matrix and the soluble lipids as resistances in series, it could be demonstrated that the soluble lipids were the main resistance for oxygen permeability in Citrus cuticles. However, in Lycopersicon and Capsicum, both the cutin matrix and the soluble lipids determined the total resistance. P values were not affected by either the proton concentration (pH 3–9) or the cations (Na⁺, Ca²⁺) present at the morphological inner side of the cuticles. It is concluded that the water content of cuticles does not affect the permeability properties for oxygen. Partition coefficients indicated a high solubility of oxygen in the cuticle of Citrus. The data suggest a solubility process in the cuticle of Citrus with respect to oxygen permeation.Abbreviations CM cuticular membrane - MX cutin polymer matrix - SCL soluble cuticular lipids     

Protecting against water loss: analysis of the barrier properties of plant cuticles

The cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other primary parts of higher plants. More than 100 mean values for water permeabilities determined with isolated leaf and fruit cuticles from 61 plant species are compiled and discussed in relation to plant organ, natural habitat and morphology. The maximum barrier properties of plant cuticles exceed that of synthetic polymeric films of equal thickness. Cuticular water permeability is not correlated to the thickness of the cuticle or to wax coverage. Relationships between cuticular permeability, wax composition and physical properties of the cuticle are evaluated. Cuticular permeability to water increases on the average by a factor of 2 when leaf surface temperature is raised from 15 degrees C to 35 degrees C. Organic compounds of anthropogenic and biogenic origin may enhance cuticular permeability. The pathway taken by water across the cuticular transport barrier is reviewed. The conclusion from this discussion is that the bulk of water diffuses as single molecules across a lipophilic barrier while a minor fraction travels along polar pores. Open questions concerning the mechanistic understanding of the plant cuticular transport barrier and the role the plant cuticle plays in ensuring the survival and reproductive success of an individual plant are indicated.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Water permeability of plant cuticles

Using the system vapor/membrane/liquid, permeability coefficients of cuticular transpiration (P _ct) were determined as functions of water activity in the vapor (a _wv). Enzymatically isolated cuticular membranes (CM) of Citrus aurantium L. and nonisolated CM of onion bulb scales and eggplant fruits were investigated. P _ct of Citrus and eggplant CM decreased with decreasing a _wv, while permeability coefficients of CM of onion were independent of a _wv. Extraction of soluble cuticular lipids (SCL) from the CM of Citrus increased permeability coefficients by a factor of approximately 500. This extraction had no effect on the dependence of P _ct on a _wv.Treating cuticular membranes as a resistance network consisting of SCL and the polymer matrix, it is shown that the permeability of onion CM is determined by the resistance of the SCL arranged in series with the polymer matrix. In this type of CM liquid and vapor are separated by a continuous, nonporous layer of SCL, and the driving force of transpiration is the gradient of partial pressure of water vapor across the SCL layer. In the CM of Citrus and eggplant, the SCL layer is traversed by polar pores that swell or shrink depending on a _wv. However, liquid continuity is maintained across these membranes down to a _wv=0.22, the lowest value used. In this type of membrane the driving force of transpiration is the water potential gradient across the membrane.Abbreviations CM cuticular membrane - MX polymer matrix - SCL soluble cuticular lipids - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES (N-morpholino)ethane sulfonic acid - SADH succinic acid 2,2-dimethyl hydrazide     

Mechanical hysteresis of insect cuticles

Solid cuticles exhibit cyclic-hardening whereas intersegmental membranes undergo stress-softening on tensile mechanical hysteresis. These differences are related to the arrangement of chitin microfibrils with respect to direction of the applied load and to the nature and extent of matrix stabilization. It is suggested that cyclic-hardening provides a fine control for intra-instar larval growth in some caterpillars. In addition, it may also provide skeletal regidity on ecdysis before completion of bulk tanning of the presumptive exocuticle in more typical examples of solid cuticle.     

The use of biological stains in the analysis of late Palaeozoic pteridosperm cuticles

The use of biological stains in the cuticular analysis of late Palaeozoic pteridosperms based on specimens from the Stephanian Blanzy-Montceau Basin (Central France) is discussed. Bismarck Brown, Malachite Green G, Methylene Blue, Methyl Green, Neutral Red, Safranin T, and a double staining with Neutral Red and Malachite Green G, were tested. Bismarck Brown, Malachite Green G, Methylene Blue, and Neutral Red increase contrast and emphasize differences in cutinization. The double staining in Neutral Red and Malachite Green G enhances the three-dimensional morphology of complex epidermal structures. Safranin T increases contrast, emphasizes cutinization differences, and enhances the three-dimensional morphology of complex epidermal features. The colour photography of cuticles is normally not affected by the presence of stains, but some stains mask black-and-white half-tones.     

The in vivo and in vitro interactions of elastic and rigid vesicles with human skin

Elastic vesicles are the most novel development in vesicular systems design for dermal and transdermal drug delivery. However, interactions between these vesicles and human skin are not yet fully understood. In this study, the in vivo and in vitro interactions between elastic-, rigid vesicles and micelles with human skin were investigated. Vesicle and micelle solutions were applied onto human skin in vitro and in vivo. Subsequently, a series of tape strippings were performed, which were visualised by freeze fracture electron microscopy (FFEM). The results showed no ultrastructural changes in skin treated with rigid vesicles. Skin treated with elastic vesicles, however, showed a fast partitioning of intact vesicles into the deeper layers of the stratum corneum (SC), where they accumulated in channel-like regions. Only little vesicle material was found in the deepest layers of the SC, suggesting that the partitioning of intact vesicles from the SC into the viable epidermis is unlikely to happen. Treatment with micelles resulted in rough, irregular fracture planes. Similar results were obtained in vitro and in vivo, indicating an excellent in vitro/in vivo correlation. These results support the hypothesis that elastic vesicles have superior characteristics to rigid vesicles for the interaction with human skin. Elastic vesicles and micelles demonstrated very different interactions with human skin and hence probably also have different mechanisms of action for the enhancement of drug transport.     

The cuticles of some Angiosperm leaves and fruits

The cuticles of twenty-four species from a wide range of mono- and di-cotyledonous plants were examined by chemical methods. The cuticles differ markedly in the amount and composition of the surface wax, in the thickness of the cuticular membrane, and in the content and composition of the cutin of the membrane. Fruits usually have heavier wax deposits and much thicker membranes than leaves. No direct relationship exists between surface waxiness and thickness of the membrane. Alkanes and primary alcohols are prominent in many of the surface waxes; triterpenoids occur less frequently. The cutin content of the membrane varies considerably; a delicate membrane tends to have a low content of cutin in which fatty acids are prevalent, and a well-developed membrane a higher content of cutin more rich in hydroxy-fatty acids. 10,16-Dihydroxyhexadecanoic acid is often an important constituent of cutin; 9,10,18-trihydroxyoctadecanoic acid is most prominent in the cutin of thicker membranes. The possible influence of the variations in cutin acids upon the structure of cutin and the taxonomic implications of wax and cutin composition are discussed.     

Phenolic constituents of tomato fruit cuticles

Chalconaringenin, naringenin, naringenin-7-glucoside, and m- and p-coumaric acids have been identified in the fruit cuticles of three tomato cultivars. The phenolic content of the cuticles increased substantially during fruit development, those from immature green and mature ripe fruits of cv Ailsa Craig yielding respectively 2.8 and 61 μg/cm² (representing 1.4 and 6% of the total membrane wt). Coumaric acids, present only in the ‘cutin-bound’ phenolics, increased from 2 to 24 μg/cm² during fruit development. Flavonoids, synthesized mainly during the climacteric, occurred free in the epicuticular (0.3–7.2 μg/cm²) and cuticular (0.7–5.7 μg/cm²) phenolics but the major part of this class of constituents in ripe fruit cuticles was also ‘bound’ to the cutin matrix (30–43 μg/cm²). The composition of the flavonoid fraction was controlled by the spectral quality of incident radiation, red light favouring the formation of chalconaringenin.     

The structure and lamination of some arthropod cuticles

The cuticles of some insectan, chelicerate, and crustacean arthropods have been examined, and evidence obtained of the presence in the laminae of material of different composition from the remainder of the cuticle. This material is present both between the layers of crossed fibres of the balken type of cuticle, and in the plumose type of lamina. It has been observed in some instances to form a clearly defined membrane.     

The consequences of predation in the population biology of the monocarpic species Cirsium palustre and Cirsium vulgare

Summary Predation by rabbits, Cheilosia grossa and especially Epiblema scutulana considerably reduces achene production in Cirsium palustre and Cirsium vulgare. In both species larger plants in a population are more frequently attacked, however the effect of predation is greater in medium sized plants. As a result the contribution of larger plants to the total achene production of the population is increased.In Cirsium vulgare larger populations are more susceptible to attack. The pattern and consequences of predation are discussed in relation to the occurrence in temporary habitats and to selectional processes. Predation intensifies existing developments in the population biology of these species. To compare the effect of predation for both species, detailed information of their population biology is necessary. At the moment the outcome of the comparison can only be indicated.Publication of the Meijendel-comité, Nieuwe Serie no. 62